USER GUIDE
SuperScript® Choice System for
cDNA Synthesis
Catalog number 18090-019
Document Part Number18090
Publication Number MAN0000420
Revision 3.0
For Research Use Only. Not for use in diagnostic procedures.
Table of Contents
1. Notices to Customer..................................................................................1
1.1Precautions.........................................................................................................1
1.2 Limited Label License No. 358: Research Use Only........................................1
1.3Trademarks.........................................................................................................1
1.4Disclaimer...........................................................................................................1
2.Overview.........................................................................................................2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
cDNA Libraries....................................................................................................2
mRNA Isolation...................................................................................................2
Choosing the Priming Method...........................................................................4
First-Strand Synthesis........................................................................................5
Second-Strand Synthesis..................................................................................6
Maximizing Ligation Efficiency by Adapter Addition..........................................6
Size Fractionation of cDNA................................................................................7
Choosing the Cloning Vector.............................................................................7
2.8.1 Plasmid Vectors..................................................................................... 8
2.8.2
λ Vectors................................................................................................ 8
Ligation of Size-Fractionated cDNA to the Vector of Choice..........................10
3.Methods.........................................................................................................11
3.1Components.....................................................................................................11
3.2 General Comments..........................................................................................11
3.2.1 mRNA Purification................................................................................11
3.2.2 Advance Preparations..........................................................................12
3.2.3 Time Planning.......................................................................................12
3.2.4 Utilization of Reagents..........................................................................12
3.3 First-Strand Synthesis......................................................................................14
3.4 Second-Strand Synthesis................................................................................16
3.5
EcoR I (Not I) Adapter Addition........................................................................17
3.6 Phosphorylation of EcoR I-Adapted cDNA......................................................17
3.7 Column Chromatography.................................................................................17
3.8 Ligation of cDNA to a Plasmid Vector..............................................................21
3.9 Ligation of cDNA to λgt11 and λgt10 Vectors .................................................21
3.10 Ligation of cDNA to a λZipLox Vector..............................................................22
3.11 Analysis of cDNA Products..............................................................................22
3.11.1 First-Strand Yield.................................................................................22
3.11.2 Second-Strand Yield............................................................................23
3.11.3 Gel Analysis.........................................................................................23
3.12 Analysis of cDNA from the cDNA Size Fractionation Column........................24
4.Troubleshooting........................................................................................26
4.1 Isolation of mRNA.............................................................................................26
4.2 First-Strand Reaction.......................................................................................26
4.3 Second-Strand Reaction..................................................................................27
4.4
EcoR I (Not I) Adapter Addition........................................................................27
4.5 Phosphorylation of EcoR I-Adapted cDNA......................................................27
i
Table of Contents
4.6 Column Chromatography.................................................................................28
4.7 Ligation of cDNA to a Plasmid Vector..............................................................28
4.8 Ligation of cDNA to a λ Vector.........................................................................28
5.References...................................................................................................29
6. Related Products.......................................................................................30
Figures
1.
2.
3.
4.
5.
6.
ii
Summary of the SuperScript® Choice System Procedure.........................................3
Effect of Random Hexamer Concentration on First-Strand cDNA Synthesis............4
Sequence of the EcoR I (Not I) Adapter......................................................................7
Detailed Protocol Flow Diagram................................................................................13
Alkaline Agarose Gel Analysis of First and Second-Strand
cDNA Synthesized with the SuperScript® Choice System.......................................24
Electrophoretic Analysis of Size-Fractionated cDNA................................................25
1
Notices to Customer
1.1Precautions
Warning: This product contains hazardous reagents. Consult the applicable SDS(s)
before using this product. Disposal of waste organics, acids, bases, and radioactive
materials must comply with all appropriate federal, state, and local regulations.
1.2 Limited Label License No. 358: Research Use Only
The purchase of this product conveys to the purchaser the limited, non-transferable
right to use the purchased amount of the product only to perform internal research
for the sole benefit of the purchaser. No right to resell this product or any of its
components is conveyed expressly, by implication, or by estoppel. This product is
for internal research purposes only and is not for use in commercial applications of
any kind, including, without limitation, quality control and commercial services such
as reporting the results of purchaser's activities for a fee or other form of
consideration. For information on obtaining additional rights, please contact
[email protected] or Out Licensing, Life Technologies, 5791 Van Allen
Way, Carlsbad, California 92008.
1.3Trademarks
The trademarks mentioned herein are the property of Life Technologies Corporation
or their respective owners.
Sephacryl is a registered trademark of GE Healthcare Bio-Sciences.
TRIzol is a registered trademark of Molecular Research Center, Inc.
RNase Away and ART are registered trademarks of Molecular Bio-Products, Inc.
1.4Disclaimer
LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL
WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED,
INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR
A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED
BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE
LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR
ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE
OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS
DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
©2013 Life Technologies Corporation. All rights reserved.
1
2
Overview
2.1 cDNA Libraries
A cDNA library is an array of DNA copies of an mRNA population that are
propagated in a cloning vector and usually maintained in E. coli. A good cDNA
library is large enough to contain representatives of all sequences of interest, some
of which may be derived from low-abundance mRNAs. Depending on the research
objective, cDNA library construction can begin by priming first-strand synthesis with
oligo(dT) or with random hexamers. These two priming methods will ultimately
produce two different types of cDNA libraries, each of which will serve a different
purpose. Libraries produced using oligo(dT) priming will contain cDNA inserts of the
largest size; libraries produced using random hexamer priming may contain a
higher proportion of cDNA with the 5´-most sequence information and may include
cDNA copies of poly(A)- as well as poly(A)+ RNA. Careful planning and appropriate
choices will result in cDNA libraries tailored to specific research objectives.
Because a cDNA library is the end product of many individual steps, its quality can
be compromised by inefficiency at any point in the procedure. The SuperScript®
Choice System integrates state-of-the-art cDNA synthesis with simplified
downstream technology to produce cDNA that can be ligated to any
EcoR I-digested vector for subsequent introduction into E. coli. If the starting mRNA
is of high quality, the cDNA library constructed with this system will satisfy the
preceding criteria.
cDNA libraries can be broadly classified as directional or random. Whereas all
members of a directional library contain cDNA inserts cloned in a specific
orientation relative to the transcriptional polarity of the original mRNAs, members of
random libraries contain cDNA inserts cloned in either orientation. For maximum
versatility, the SuperScript® Choice System has been designed for generation of
random libraries: the double-stranded, EcoR I-ended cDNAs produced with this
system are suitable for insertion into the vast majority of existing vectors.
The major steps in constructing a random cDNA library from an mRNA population
using the SuperScript® Choice System are summarized in Figure 1.
2.2 mRNA Isolation
Construction of a good cDNA library begins with the preparation of high-quality
mRNA. The quality of the mRNA dictates the maximum amount of sequence
information that can be converted into cDNA. Thus, it is important to optimize the
isolation of mRNA from a given biological source and to prevent adventitious
introduction of RNases into a preparation that has been carefully rendered RNasefree. For optimal results, the mRNA must be purified over an affinity column
[oligo(dT) cellulose being the most commonly used matrix] to select the
polyadenylated [poly(A)+] RNA (1). Since the vast majority of mRNA is poly(A)+, this
selection operationally defines the mRNA population. Typically, 0.5% to 2% of a
total RNA population is mRNA, so isolation of this fraction from rRNA, tRNA, and
degraded mRNA enhances the synthesis of first-strand cDNA and minimizes
spurious transcription of non-mRNAs. An mRNA preparation that has undergone
two selections over this matrix (in which the eluate from one round of purification
has been bound to the column and eluted a second time) will produce the highest
quality mRNA. When properly prepared, oligo(dT) cellulose-purified RNA will be
≥90% mRNA.
2
2
Figure 1. Summary of the SuperScript® Choice System procedure.
The amount of mRNA needed to prepare a library is dependent on the efficiency of
the individual steps needed to convert the mRNA into a form that can be cloned and
on the efficiency with which the recombinant molecules can be introduced into a
host. Generally, 1 to 5 µg of mRNA will be sufficient to construct a cDNA library
containing 106 to 107 clones in E. coli.
3
Overview
2.3 Choosing the Priming Method
First-strand cDNA synthesis is most commonly primed using oligo(dT) or
modifications of this sequence (such as primer-adapters) that bind to the poly(A) tail
of mRNA. This priming method offers two major advantages: only poly(A)+ RNA is
copied, and most cDNA clones begin at the 3´ terminus of the mRNA. At the same
time, oligo(dT) priming has certain limitations: some cDNA clones may not be full
length, due to RNA secondary structure or pausing by reverse transcriptase, and
poly(A)- mRNA cannot be copied.
An alternative method is to use random hexamers, which, in theory, are capable of
binding and priming throughout virtually any RNA template. Random hexamers,
which may be used either by themselves or in combination with oligo(dT), have
been instrumental in producing cDNAs containing more 5´ information than those
primed with oligo(dT) alone (2,3). In addition, random hexamers can be used to
generate cDNA libraries from poly(A)- mRNA (4) and single-stranded viral RNAs
(2,5).
Although increasing the random hexamer concentration increases the percentage
yield of first-strand cDNA synthesis, it also decreases the average cDNA size. A
typical random hexamer titration profile (using cDNA synthesized from HeLa
mRNA), as compared to the result obtained with oligo(dT) priming, is shown in
figure 2. Identical random hexamer titration profiles are obtained whether the firststrand cDNA reaction is incubated at 37°C or 45°C, using from 1–5 µg of mRNA per
reaction, and in the presence or absence of oligo(dT) primers. Note: A certain
amount of self-priming by subpopulations of mRNA may occur in the absence of any
exogenous primers (see lane 2 of figure 2), which can contribute nominally (~6%) to
the overall first-strand cDNA yield produced by random hexamer priming.
1
2
3
4
5
6
7
|
5
|
20
|
60
|
150
|
400
8
9
Kb
12.2—
7.1—
5.1—
4.0—
3.0—
2.0—
1.6—
1.0—
0.5—
|
1 µg
|
0
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1,000 5,000
Figure 2. Effect of random hexamer concentration on first-strand cDNA synthesis.
2 µg of HeLa mRNA was primed with 1 µg of oligo(dT)12-18 (lane 1) or various amounts of
random hexamers (lanes 2 through 9).
4
The SuperScript® Choice System includes both oligo(dT)12-18 and random hexamer
primers. In the decision to use either priming method separately, or both in
combination, to synthesize cDNA, the following considerations should be noted:
1. Priming with oligo(dT) by itself is decidedly better at producing larger cDNA
inserts.
2. Random hexamer priming yields cDNA that are smaller on average but that
may better represent the entire RNA template. Priming with random hexamers
at a concentration of 50–150 ng per reaction generally yields twice the amount
of 5´-end information of the β-actin mRNA as oligo(dT) priming (6). For some
RNAs, however, higher concentrations of random hexamers may be needed to
increase the proportion of cDNA containing the 5´-most sequence information.
3. Random hexamers and oligo(dT), when used in combination, should be added
simultaneously to ensure all possible priming events.
2
2.4 First-Strand Synthesis
Avian myeloblastosis viral (AMV) reverse transcriptase (RT) was the first enzyme
used to synthesize cDNA in vitro, and much of the early work in cDNA synthesis
and cloning was developed using this enzyme. However, the successful cloning of
the Moloney murine leukemia virus (M-MLV) RT (7) has provided researchers with
an alternative enzyme. The cloned M-MLV RT gene has been further engineered to
produce a novel enzyme (SuperScript® II RT) with reduced RNase H activity (8).
This modification is significant because RNase H activity is detrimental to the firststrand cDNA synthesis reaction in two ways:
1. The initiation of first-strand synthesis depends upon the hybridization of a
primer to the mRNA, usually at the poly(A) tail. This hybrid is a substrate, not
only for the polymerase activity of the RT but also for the RNase H activity (9).
In the resulting competition between these two activities, the extent to which
the RNase H activity destroys the hybrid prior to the initiation of polymerization
determines the maximal number of initiation events that can actually occur.
Hydrolysis of the RNA in the hybrid reduces the maximal yield of cDNA by
effectively removing a portion of the mRNA from the reaction.
2. When the RT is synthesizing the first-strand cDNA, the RNase H activity will
quickly degrade the template that has already been copied because the
mRNA is in hybrid form as a result of the polymerization reaction. If the
scissions in the mRNA occur near the point of chain growth, the uncopied
portion of the mRNA can dissociate from the transcriptional complex, resulting
in termination of cDNA synthesis for that template and consequent reduction in
the yield of full-length cDNA. This problem can be exacerbated if the RT
pauses during transcription at certain primary or secondary structural domains.
When used with synthetic RNA produced in vitro, SuperScript ® II RT has
demonstrated significantly greater full-length cDNA synthesis and higher yields of
first-strand cDNA than other commercially available RTs (10, 11, 12).
The reaction conditions for first-strand synthesis catalyzed by SuperScript® II RT
have been optimized for yield and size of the cDNAs. The optimal first-strand
reaction temperature for SuperScript® II RT is 37°C; however, should secondary
structure make reverse transcription difficult, a higher reaction temperature may be
used. SuperScript ® II RT is stable at 45° to 50°C and can be used at this
temperature, if necessary.
The amount of mRNA can be as high as 5 µg in a 20-µL first-strand cDNA synthesis
reaction. We recommend using at least 1 µg of mRNA so that there will be sufficient
material at the end of the procedure to obtain the required number of clones. The
amount of SuperScript® II RT needed in the first-strand reaction varies linearly with
the amount of mRNA: 200 units of SuperScript® II RT for ≤1 µg of mRNA, and
200 units per µg for 1–5 µg of mRNA. Although the exact ratio of SuperScript® II RT
to mRNA is not critical, these approximate proportions have produced reliable
results.
5
Overview
2.5 Second-Strand Synthesis
The primary sequence of the mRNA is recreated as second-strand DNA using the
first-strand cDNA as a template. The SuperScript ® Choice System uses nick
translational replacement of the mRNA to synthesize the second-strand cDNA. First
described by Okayama and Berg (13), and later popularized by Gubler and Hoffman
(14), second-strand synthesis is catalyzed by E. coli DNA polymerase I in
combination with E. coli RNase H and E. coli DNA ligase. Although RNase H is not
essential if the first-strand synthesis is catalyzed by AMV or M-MLV RT, E. coli
RNase H must be included in the second-strand reaction when SuperScript® II RT
has been used for first-strand cDNA synthesis. E. coli DNA ligase has been shown
to improve the cloning of double-stranded (ds) cDNA synthesized from longer
(≥2 kb) mRNAs (15).
The first and second-strand syntheses are performed in the same tube without
intermediate organic extraction or ethanol precipitation. This one-tube format
speeds the synthesis procedure and maximizes recovery of cDNA. The efficiency of
the second-strand reaction is influenced by the amount and concentration of the
reactants, so the instructions must be followed as described for best results. The
second-strand reaction is incubated at 16°C to prevent spurious synthesis by DNA
polymerase I due to its tendency to strand-displace (rather than nick translate) at
higher temperatures. The last step in the cDNA synthesis procedure is to ensure
that the termini of the cDNA are blunt. This is easily done by adding T4 DNA
polymerase to the second-strand reaction mixture and incubating briefly at 16°C.
The cDNA is then deproteinized by organic extraction and precipitated with ethanol
to render it ready for downstream manipulation.
2.6 Maximizing Ligation Efficiency by Adapter Addition
The product of the first and second-strand synthesis reactions performed using the
SuperScript® Choice System is blunt-ended cDNA, a poor substrate for T4 DNA
ligase. To maximize ligation efficiency into the vector, the blunt ends of the cDNA
are converted to termini that contain 5´ extensions by adding adapters to the cDNA.
Adapters are short, duplex oligomers, blunt-ended at one terminus and containing a
4-base, 5´ extension at the other terminus. The blunt-end ligation of the adapters to
the cDNA can be driven by adapter excess, much the way molecular linkers are
added to DNA. However, unlike linkers, adapters contain preformed extensions and
do not require restriction digestion to expose the termini.
The 4-base, 5´ extension of the adapters provided with the SuperScript® Choice
System corresponds to the termini produced by digestion with EcoR I. The
sequence of the EcoR I (Not I) adapter included in the SuperScript® Choice System
is shown in Figure 3. Several details should be noted:
1.
The recognition sequences for Not I and Sal I are contained within the EcoR I
(Not I) adapter to allow easy release of cDNA inserts. Both restriction
endonuclease sites are extremely rare in mammalian genomes, occurring
approximately once in 106 bp (Not I) and 105 bp (Sal I).
2. Only one of the oligomers of the EcoR I (Not I) adapter is phosphorylated,
which drives adapter-to-cDNA ligation and essentially eliminates cDNA-tocDNA ligation.
6
5´-AATTCGCGGCCGCGTCGAC-3´
3´GCGCCGGCGCAGCTGp-5´
EcoR I
Not I
Sal I
2
Figure 3. Sequence of the EcoR I (Not I) adapter.
Adding the EcoR I (Not I) adapters to the cDNA places the same EcoR I
5´ extension at both ends of the cDNA. The cDNA is then used to construct a
random library by ligating it to an EcoR I-digested vector that has been
dephosphorylated to reduce the background arising from self-ligation of the vector.
This, in turn, requires phosphorylation of the adapted cDNA at its 5´ termini so that it
can be ligated to the 5´-dephosphorylated, EcoR I-digested vector. The ligation and
phosphorylation steps used in the SuperScript® Choice System are performed in the
same buffer without any organic extraction or ethanol precipitation, which
maximizes efficiency and facilitates cDNA recovery.
2.7 Size Fractionation of cDNA
Size fractionation of cDNA, following adapter addition and phosphorylation, is
important because residual adapters are present in large molar excess and can
impede vector ligation to cDNA by ligating to the EcoR I termini of the predigested
vector. Size fractionation also reduces the tendency of smaller (<500 bp) inserts to
predominate the library. These smaller cDNAs can arise for several reasons:
1.
Most mRNA preparations are not size-selected, so partially degraded mRNAs
can be selected on the oligo(dT) cellulose columns along with longer mRNAs.
These will be reverse transcribed into small cDNAs.
2. If extreme care is not taken to prevent RNase contamination during first-strand
synthesis, degradation can occur when the mRNA is manipulated.
3. Some mRNAs contain regions that are not readily reverse transcribed, and RT
is not able to synthesize complete first strands.
Column chromatography is the simplest method of producing size-fractionated
cDNA, free of adapters and other low molecular weight DNAs. The SuperScript®
Choice System contains three 1-mL, disposable columns, prepacked with
Sephacryl® S-500 HR, that quickly and easily remove DNAs <500 bp and sizefractionate cDNAs >500 bp, as well as perform an exchange of buffers, thus
facilitating construction of libraries from fractions enriched for larger cDNA. The
column chromatography buffer (described in Chapter 3) is formulated to allow the
cDNA in the column factions to be ligated directly into predigested plasmid cloning
vectors. Although the final yield of size-fractionated cDNA will depend upon the
recovery at each step of the procedure, the average overall yield should be
5%–10% of the mass of the starting mRNA.
2.8 Choosing the Cloning Vector
Two types of vectors are generally used for cDNA cloning in E. coli plasmids and
bacteriophage lambda (λ) derivatives. Several factors affect the choice of vector.
Ease of use. Cloning into plasmids is generally easier for the novice because it
requires less manipulation of the cDNA and avoids potential problems when
propagating phage. However, this consideration alone should not preclude using a λ
vector if it is otherwise the best choice. For detailed discussions of cDNA cloning
into λ vectors, see references 16 and 17.
7
Overview
Antibody screening. λ vectors are the better choice if the cDNA library is to be
screened with the antibody (18). Although plasmid-based cDNA libraries may also
be screened by this method (19), the process is more cumbersome and
considerably more tedious.
Nucleic acid screening. Choosing between plasmid and λ vectors is less critical if
the library will be screened with a nucleic acid probe because performance is similar
in both systems (20,21). Other considerations (such as subcloning capability) may
dictate which system to use.
The SuperScript® Choice System is designed to produce cDNA containing EcoR
I-cohesive ends suitable for ligation to any EcoR l-digested, dephosphorylated
plasmid or λ vector (consult reference 17 for details on preparing dephosphorylated
vectors).
2.8.1 Plasmid Vectors
There are many plasmid vectors available that are compatible with the SuperScript®
Choice System for cDNA Synthesis. Since the SuperScript ® Choice System is
designed to produce cDNA containing EcoR I-cohesive ends, the plasmid must
contain a unique EcoR I site within the multiple cloning site. When choosing a
plasmid consider what screening method will be used and if the library will be used
in a subtractive cDNA library construction. If the library will be used for making RNA,
in vitro translation, or subtraction procedures the multiple cloning site must be
flanked by RNA polymerase promoters (i.e. SP6, T7, or T3 RNA polymerase
promoters). A vector with an f1 origin of replication can be infected with M13K07
helper phage to produce single-strand plasmid DNA for sequencing or in vitro
mutagenesis (22). Nested deletions for sequencing are facilitated by having
restriction endonuclease sites in the multiple cloning site clustered together.
Plasmid shuttle vectors for transient expression of cloned genes in mammalian cells
such as COS cells and DNA cloning in E. coli can also be used with the
SuperScript® Choice System for cDNA Synthesis.
Plasmid pcDNA3 (+) is a multifunctional vector for cDNA cloning, in vitro
transcription, and dideoxy sequencing. The plasmid contains a unique multiple
cloning site with restriction sites for EcoR I and 11 other restriction endonucleases.
There is a T7 RNA polymerase promoter that can be used to generate RNA for
probes, in vitro translation, or subtracted cDNA libraries. The plasmid contains the
CMV promoter, for expression of cloned genes. DNA inserts can be sequenced from
double-stranded DNA using T7 forward or BGH reverse sequencing primers. Singlestranded plasmid DNA for sequencing or in vitro mutagenesis can be generated by
infection of transformed cells with an appropriate helper phage such as M13K07
(22). The β-lactamase gene on the plasmid provides for convenient selection by
ampicillin resistance.
2.8.2 λ Vectors
λgt10, EcoR I Arms is a precut version of λgt10, purified from bacteriophage DNA
(imm434 b527), which has been prepared by ligation at the cos sites, digestion with
EcoR I, and dephosphorylation. This vector contains an EcoR I site within the
repressor gene and can accommodate inserts up to 7 kb in length (16). Interruption
of the repressor gene by insertion of cDNA into the EcoR I site converts the phage
from cl+ to cl-, which changes plaque morphology from turbid to clear when plated
on an E. coli strain such as C600 (24). By using the bacterial strain C600hflA150
that contains a high frequency lysogeny mutation (16), lytic growth is repressed so
effectively that plaque formation by cl+ phage does not occur; only cl- (recombinant)
phage produce plaques, significantly reducing the background. Because λgt10 is
not an expression vector, recombinant libraries prepared in λgt10 cannot be
screened with antibodies.
8
λgt11, EcoR I Arms is a precut version of λgt11, purified from bacteriophage DNA
(lac5 cl857 nin5 Sam100), which has been prepared by ligation at the cos sites,
digestion with EcoR I, and dephosphorylation. Unlike λgt10, λgt11 is an expression
vector: libraries prepared in λgt11 can be screened immunologically using specific
antibodies (18,25), as well as with nucleic acid probes. This vector contains a
unique EcoR I cloning site near the end of a β-galactosidase coding sequence and
can accept inserts up to 7.2 kb in length. If cDNA containing an open reading frame
is inserted into this site in the correct orientation, a fusion protein is produced when
expression from the lac promoter is induced with isopropylthio-β-galactoside (IPTG).
Thus, expression of proteins, which may be toxic to the cell, can be delayed until the
library has been amplified and is ready for immunological screening. The lac
promoter is also used to control expression of the cloned gene if large-scale
synthesis is needed for purification. Insertion of cDNA into the lacZ gene of λgt11
will produce plaques that are usually colorless instead of blue on plates containing
IPTG and X-gal. The ratio of colorless to blue plaques is often used to estimate the
percentage of recombinants in the library.
2
λZipLox, EcoR I Arms is a precut version of λZipLox, which has been prepared by
ligation at the cos sites, digestion with EcoR I, and dephosphorylation. Libraries
prepared in λZipLox can be screened immunologically using specific antibodies
(18,25), as well as with nucleic acid probes. λZipLox contains the plasmid pZL1,
flanked by loxP sequences, between portions of the left and right arms of λgt10 and
λgt11. cDNA inserts cloned into the EcoR I site of λZipLox reside within the
inducible lac Z´ gene commonly found in pUC-type plasmid vectors. When the lac
promoter is induced with IPTG, the cloned gene is expressed as a fusion protein
embedded within the amino-terminal portion of the β-galactosidase fragment
encoded by lac Z´. Following cloning and selection of desired clones, the cDNA can
be recovered in the autonomously replicating multifunctional plasmid pZL1 using a
simple in vivo excision protocol, obviating the need for tedious subcloning.
Life Technologies vector sequences, restriction information, and maps can be found
in the Vector Data area of our web site www.lifetechnologies.com.
9
Overview
2.9 Ligation of Size-Fractionated cDNA to the Vector of Choice
The ligation reactions described in Sections 3.8, 3.9 and 3.10 in Chapter 3 will
suffice for most applications. We have found that 10–20 ng of cDNA saturates
50 ng of plasmid vector and that 50 ng of cDNA saturates 500 ng of λ vector; to use
more cDNA is wasteful (as little as 1 ng of cDNA can be used in either ligation
reaction). For any particular population of cDNA, however, these ratios may
not be optimal. If the described ligation conditions do not yield enough
transformants or plaques to make your library complete, the vector-to-cDNA
ratio yielding the maximum number of clones should be determined
empirically.
The yield is also dependent upon the transformation efficiency of the E. coli cells
used to plate the plasmid-based library or the efficiency of the in vitro packaging
methods used to introduce the λ-ligated cDNA into E. coli by infection. For either
type of vector, the following considerations should be noted:
10
Plasmid Vectors. If the E. coli competent cells yield ~1 × 109 transformants/µg
of pUC19 plasmid DNA, then the ligated cDNA should yield 0.5 to 1 × 107
transformants/µg of vector; this is equivalent to 2.5 to 5 × 107 transformants/µg
of cDNA. Thus, a plasmid library containing 5 × 105 clones can be constructed
from 10 ng of cDNA used in the ligation reaction in Section 3.8. Plasmidligated cDNA can also be introduced into E. coli cells by electroporation, which
generally will yield a greater number of transformants (2.5 × 108 to 1 × 109
transformants/µg cDNA) from the same amount of ligated cDNA.
Electroporation may be especially useful if the library must be very large or if
you have <10 ng of cDNA.
λ Vectors. If the in vitro packaging extract yields approximately 5 × 109 plaque
forming units (pfu)/µg of ligated, wild-type λ DNA, then the ligated cDNA
should yield 0.5 × 107 to 1 × 107 pfu/µg of vector; this is equivalent to 2.5 × 108
to 5 × 108 pfu/µg of cDNA. Thus, a library containing 2 × 106 clones can be
constructed from as little as 10 ng of cDNA.
Methods
3
3.1Components
The components of the SuperScript® Choice System for cDNA Synthesis are as
follows. Components are provided in sufficient quantities to perform three separate
experiments, each converting up to 5 µg of mRNA into size-fractionated, EcoR
I-adapted cDNA, ready for ligation into any EcoR I-digested, dephosphorylated
vector. Store chromatography columns (Part No. 8092CL) at 2°C to 8°C and store
the reagent assembly (Part No. 8090RT) at –30°C to –10°C.
ComponentAmount
Oligo(dT)12-18 primer (0.5 µg/µL)........................................................................................... 10 µL
Random Hexamers (50 ng/µL)............................................................................................ 50 µL
5X First-Strand Buffer [250 mM Tris-HCl (pH 8.3),
375 mM KCl, 15 mM MgCl2]........................................................................................... 1 mL
0.1 M DTT.......................................................................................................................... 250 µL
10 mM dNTP Mix (10 mM each dATP, dCTP, dGTP, dTTP)................................................ 20 µL
SuperScript® II RT (200 units/µL)........................................................................................ 50 µL
5X Second-Strand Buffer [100 mM Tris-HCl (pH 6.9),
450 mM KCl, 23 mM MgCl2, 0.75 mM ß-NAD+, 50 mM (NH4)2SO4]............................ 500 µL
E. coli DNA Ligase (10 units/µL).......................................................................................... 10 µL
E. coli DNA Polymerase I (10 units/µL)............................................................................... 50 µL
E. coli RNase H (2 units/µL)................................................................................................ 20 µL
T4 DNA Polymerase (5 units/µL)......................................................................................... 10 µL
5X Adapter Buffer [330 mM Tris-HCl (pH 7.6),
50 mM MgCl2, 5 mM ATP]............................................................................................. 30 µL
EcoR I (Not I) Adapters (1 µg/µL)........................................................................................ 30 µL
T4 DNA Ligase (1 units/µL)................................................................................................. 50 µL
T4 Polynucleotide Kinase (10 units/µL)............................................................................... 10 µL
5X T4 DNA Ligase Buffer [250 mM Tris-HCl (pH 7.6),
50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000]........................................ 1 mL
DEPC-treated Water........................................................................................................ 1.25 mL
Control RNA (0.5 µg/µL)...................................................................................................... 15 µL
Yeast tRNA (1 µg/µL)......................................................................................................... 100 µL
cDNA size fractionation columns.......................................................................................... three
Manual.................................................................................................................................... one
3.2 General Comments
3.2.1 mRNA Purification
One of the most important steps preceding the synthesis of cDNA and the
establishment of a library is isolation of intact mRNA. The Micro-FastTrack® 2.0
mRNA Isolation Kit allows you to isolate mRNA in a fast and convenient procedure.
Successful cDNA synthesis demands an RNase-free environment at all times, which
will generally require the same level of care used to maintain aseptic condi­tions
when working with microorganisms. Several additional guidelines should be
followed:
1.
Never assume that anything is RNase-free, except sterile pipets, centrifuge
tubes, culture tubes, and any similar labware that is explicitly stated to be
sterile.
2. Dedicate a separate set of automatic pipets for manipulating RNA and the
11
Methods
buffers and enzymes used to synthesize cDNA. All purpose pipets may be
quickly prepared for RNA use by wiping the outside surface of the pipet with
RNase AWAY® Reagent. Use barrier tips such as ART® Tips.
3. Obtain RNase-free microcentrifuge tubes or treat tubes overnight in a 0.01%
(v/v) aqueous solution of diethylpyrocarbonate (DEPC); rinse them with
autoclaved, distilled water; and autoclave them.
4. Avoid using any recycled glassware unless it has been specifically rendered
RNase-free by rinsing with 0.5 N NaOH or RNase AWAY® Reagent followed
by copious amounts of DEPC-treated, autoclaved water or other prepared
RNase-free water. Alternatively, bake glassware at 150°C for 4 hours.
5. Use reagent grade solutions that are set aside for RNA use only. The preferred
method of obtaining RNase-free solutions is to treat them with 0.01% (v/v)
DEPC followed by autoclaving. If preparing solutions containing primary
amines (such as Tris), where DEPC cannot be used, or preparing heatsensitive solutions, use RNase-free reagents, water and labware, and filter the
solution through a 0.2-µm disposable, sterile filter.
3.2.2 Advance Preparations
Before using this system, please review the protocol flow diagram in figure 4. You
will need the following items not included in this system:
• autoclaved 1.5-mL microcentrifuge tubes
• microcentrifuge capable of generating a relative centrifugal force of 14,000 × g
• automatic pipets capable of dispensing 1 to 20 µL, 20 to 200 µL, and 200 µL
to 1 mL
• autoclaved, disposable tips for automatic pipets
• disposable gloves
• 16°C and 37°C water baths
•1–10 µCi [α-32P]dCTP (400 to 3,000 Ci/mmol)
• 500 mL 10% (w/v) TCA (trichloroacetic acid)
Note: The use of pre-made phenol:
chloroform:isoamyl alcohol (25:24:1,
v/v) is recommended. If making your
own, saturate the redistilled phenol
with TEN buffer, not with distilled
water.
Note: Suggested stopping points are
noted in the protocol with the
icon.
12
containing 1% (w/v) sodium
pyrophosphate (store at 4°C)
• glass fiber filters (1 × 2 cm) (Whatman GF/C or equivalent)
• buffer-saturated phenol:CHCl3:isoamyl alcohol [25:24:1 (v/v/v)]
• TEN buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 25 mM NaCl; autoclaved]
• 7.5 M ammonium acetate (NH 4OAc) filtered through a sterile, 0.2-µm
nitrocellulose filter
• 70% (v/v) ethanol (–20°C)
3.2.3 Time Planning
Starting with poly(A)+ RNA, these protocols are designed to yield cDNA containing
EcoR I-cohesive ends in ~2 days. For best results, the procedure should be
completed as quickly as possible because radiochemical effects induced by the
decay of the 32P in the cDNA can diminish transformation efficiencies over time.
We recommend that the protocols be completed as follows:
Day 1: Sections 3.3 and 3.4, and steps 1 and 2 of Section 3.5 (overnight
incubation of EcoR I (Not I) Adapter Addition reaction).
Day 2: Sections 3.5 through 3.8 or 3.9.
If interrupting the procedure at any other point becomes necessary, you may do so
following Sections 3.4 or 3.7. When stopping at any such point, always store the
cDNA as the uncentrifuged ethanol precipitate at –20°C to minimize the
aforementioned effects of 32P decay.
3.2.4 Utilization of Reagents
Reagents included with the SuperScript® Choice System are provided in sufficient
quantities to perform three complete experiments converting up to 5 µg of mRNA
into size-fractionated, EcoR I-adapted cDNA. Additionally, the components for first
3
Poly(A)+ mRNA
70°C, 10 min
37°C, 2 min
Oligo(dT)12-18, Random Hexamers,or both
DEPC-Treated Water
5X First-Strand Buffer4
0.1 M DTT
10 mM dNTP Mix
[α-32P]dCTP
SuperScript® II RT
First-strand reaction
37°C, 1 h
Remove aliquot for yield
and gel electrophoretic
analyses
Transfer to ice
First-strand cDNA
DEPC-treated water
5X Second-Strand Buffer
10 mM dNTP Mix
E. coli DNA Ligase
E. coli DNA Polymerase I
E. coli RNase H
Second-strand reaction
16°C, 2 h
T4 DNA Polymerase
16°C, 5 min
Extract, precipitate
ds cDNA
DEPC-Treated Water
5X Adapter Buffer
EcoR I (Not I) Adapters
0.1 M DTT
T4 DNA Ligase
16°C, 16 h
70°C, 10 min
EcoR I-adapted cDNA
37°C, 30 min
70°C, 10 min
T4 Polynucleotide Kinase
Column chromatography
Size-fractionated cDNA
Figure 4. Detailed protocol flow diagram.
13
Methods
and second-strand synthesis are provided in sufficient quantities to perform
Sections 3.3 and 3.4 five times. You may wish to use these extra quantities to test a
small amount of your mRNA by determining first or second-strand yield and
visualizing the distribution of the products by gel electrophoresis. Alternatively, the
extra components can be held as a backup in case of accidental loss of material or
procedural error.
3.3 First-Strand Synthesis
Note: The efficiency of the second-strand
reaction is influenced by the amount and
concentration of the reactants, so the
instructions must be followed as described
for best results.
The 20-µL reaction described is designed to convert up to 5 µg of mRNA into firststrand cDNA. The amount of SuperScript® II RT added to the reaction will be
dependent upon the amount of starting mRNA. We recommend 200 units of
SuperScript® II RT for ≤1 µg of mRNA and 200 units/µg of mRNA for 1–5 µg of
mRNA.
If second-strand cDNA is to be labeled instead of first-strand cDNA, the first-strand
reaction should be set up without [α-32P]dCTP (adjust the amount of water in the
reaction to maintain the 20-µL final volume), and the reaction should be carried
through the second-strand synthesis procedure as described in Section 3.4. In this
case, add 1 µL (10 µCi/µL) [α-32P]dCTP to the second-strand reaction after the
10 mM dNTP mix is added.
A single-transcript control RNA is included in the SuperScript® Choice System as an
aid in verifying the first-strand reaction. If you decide to use the control RNA, simply
substitute 4 µL (2 µg) in the first-strand reaction for your mRNA.
Note: Do not proceed with this protocol
until you have made the appropriate
decisions regarding your choice of primer.
For more information, see Section 2.3,
Choosing the Priming Method.
1.Perform one of the following substeps – a, b, or c – depending on your
choice of priming method.
a. For priming with oligo(dT): Add 2 µL of Oligo(dT)12-18 primer to a sterile
1.5-mL microcentrifuge tube. Add mRNA, diluted as needed with DEPCtreated water, according to the following table:
µg of mRNA
≤1
2
3
4
5
mRNA (plus DEPC-treated water)
9
8
7
6
5
to a total volume (µL)
11
10
9
8
7
b.
For priming with random hexamers: Add 1 to 3 µL (50 to 150 ng) of
Random Hexamers to a sterile 1.5-mL microcentrifuge tube. Add mRNA,
diluted as needed with DEPC-treated water, according to the following
table:
µg of mRNA
≤1
2
3
4
5
mRNA (plus DEPC-treated water)
to a total volume (µL)
c.
11
10
9
8
7
For priming with both Oligo(dT) and Random Hexamers: Add 2 µL of
Oligo(dT)12-18 Primer and 1 to 3 µL (50 to 150 ng) of Random Hexamers to
a sterile 1.5-mL microcentrifuge tube. Add mRNA, diluted as needed with
DEPC-treated water, according to the following table:
µg of mRNA
≤1
2
3
4
5
14
mRNA (plus DEPC-treated water)
to a total volume (µL)
11
10
9
8
7
2. Heat the mixture to 70°C for 10 minutes and quick-chill on ice. Collect the
contents of the tube by brief centrifugation and add the following:
Component
5X First-Strand Buffer
0.1 M DTT
10 mM dNTP Mix
[α-32P]dCTP (1 µCi/µL)
Volume (µL)
4
2
1
1
3
The total volume should now correspond to the following table:
Total volume (µL)
µg of mRNA (from step 1)
≤1
2
3
4
5
19
18
17
16
15
3.Mix the contents of the tube by gently vortexing and collect the reaction by brief
centrifugation. Place the tube at 37°C for 2 minutes to equilibrate the
temperature.
4. Add SuperScript® II RT according to the following table:
SuperScript® II RT (µL)
µg of mRNA (from step 1)
≤1
2
3
4
5
1
2
3
4
5
Mix gently and incubate at 37°C for 1 hour. Regardless of the amount of
starting mRNA, the total volume should now be 20 µL.
Final composition of the reaction:
50 mM Tris-HCl (pH 8.3)
75 mM KCl
3 mM MgCl2
10 mM DTT
500 µM each dATP, dCTP, dGTP, and dTTP
50 µg/mL oligo(dT) 12-18 primer and/or 2.5–7.5 µg/mL random
hexamers
≤5 µg (≤250 µg/mL) mRNA
10,000–50,000 units/mL SuperScript® II RT
5. Place the tube on ice to terminate the reaction.
6. Remove 2 µL from the reaction and add it to a microcentrifuge tube containing
43 µL of 20 mM EDTA (pH 7.5) and 5 µL of Yeast tRNA. This mixture will be
used in calculating first-strand yield.
7. Take the remaining 18 µL of the first-strand reaction and continue immediately
with the first two steps of the second-strand reaction as described in Section
3.4.
8. While the second-strand reaction is incubating, spot duplicate 10-µL aliquots
from the diluted sample from step 6 of this section onto glass fiber filters. Dry
one of the filters under a heat lamp or at room temperature. This filter will be
used to determine the specific activity of the dCTP reaction.
9. Wash the other filter three times in sequence, for 5 minutes each time, in a
beaker containing 50 mL of fresh, ice-cold 10% (w/v) TCA containing 1% (w/v)
sodium pyrophosphate. Wash the filter once with 50 mL of 95% ethanol at
room temperature for 2 minutes. Dry the filter under a heat lamp or at room
temperature. This filter will be used to determine the yield of first-strand cDNA.
10. Count both filters in standard scintillant to determine the amount of 32P in the
reaction, as well as the amount of 32P that was incorporated. See Section 3.11,
Analysis of cDNA Products, for information needed to convert the data into
yield of first-strand cDNA.
15
Caution: If the first-strand cDNA was
labeled, the supernatant(s) will be
radioactive. Dispose of this material
properly.
11. Precipitate the remaining 30 µL of the sample from step 6 of this section by
adding 15 µL of 7.5 M NH4OAc, followed by 90 µL of absolute ethanol (–20°C).
Vortex the mixture thoroughly and immediately centrifuge at room temperature
for 20 minutes at 14,000 × g.
12. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70%
ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the
supernatant.
13. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and
proceed to Section 3.11, Analysis of cDNA Products.
3.4 Second-Strand Synthesis
This protocol is suitable for synthesizing second-strand cDNA from ≤5 µg mRNA
originally in the 20-µL first-strand reaction.
Caution: If the first or second-strand
cDNA was labeled, the supernatant(s) will
be radioactive. Dispose of this material
properly.
16
1. On ice, add the following reagents, in the order shown, to the first-strand
reaction tube:
Component
Volume (µL)
DEPC-Treated Water.......................................................................................93
5X Second-Strand Buffer.................................................................................30
10 mM dNTP Mix...............................................................................................3
E. coli DNA Ligase (10 units/µL)........................................................................1
E. coli DNA Polymerase I (10 units/µL).............................................................4
E. coli RNase H (2 units/µL)..............................................................................1
Final volume..................................................................................................150
Final composition of the reaction:
25 mM Tris-HCl (pH 7.5)
100 mM KCl
5 mM MgCl2
10 mM (NH4)2SO4
0.15 mM ß-NAD+
250 µM each dATP, dCTP, dGTP, dTTP
1.2 mM DTT
65 units/mL DNA ligase
250 units/mL DNA polymerase I
13 units/mL RNase H
2. Vortex the tube gently to mix and incubate the completed reaction for 2 hours
at 16°C. Do not let the temperature rise above 16°C.
3. Add 2 µL (10 units) of T4 DNA Polymerase and continue incubating at 16°C for
5 minutes.
4. Place the reaction on ice and add 10 µL of 0.5 M EDTA. Note: If [α-32P]dCTP
was added to the second-strand reaction, remove 10 µL from the reaction, and
add it to a microcentrifuge tube containing 35 µL of 20 mM EDTA (pH 7.5) and
5 µL of Yeast tRNA. This mixture will be used in calculating second-strand
yield. Then proceed as described in steps 8 to 10 in Section 3.3 for processing
and counting the filters.
5. Add 150 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly,
and centrifuge at room temperature for 5 minutes at 14,000 × g to separate the
phases. Carefully remove 140 µL of the upper, aqueous layer, and transfer it to
a fresh 1.5-mL microcentrifuge tube.
6. Add 70 µL of 7.5 M NH4OAc, followed by 0.5 mL of absolute ethanol (–20°C).
Vortex the mixture thoroughly and immediately centrifuge at room temperature
for 20 minutes at 14,000 × g.
7. Remove the supernatant carefully and overlay the pellet with 0.5 mL of 70%
ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the
supernatant.
8. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and
proceed to Section 3.5.
3.5 EcoRI (Not I) Adapter Addition
1.
Add the following reagents on ice, in the order shown, to the cDNA from step 8
of Section 3.4.
Component
Volume (µL)
DEPC-treated Water........................................................................................18
5X Adapter Buffer............................................................................................10
EcoR I (Not I) Adapters...................................................................................10
0.1 M DTT..........................................................................................................7
T4 DNA Ligase..................................................................................................5
Final volume....................................................................................................50
Final composition of the reaction:
66 mM Tris-HCl (pH 7.6)
10 mM MgCl2
1 mM ATP
14 mM DTT
200 µg/mL EcoR I (Not I) adapters
100 units/mL T4 DNA ligase
2. Mix gently and incubate the reaction at 16°C for a minimum of 16 hours. For
greatest convenience, simply let the reaction proceed overnight.
3. Heat the reaction at 70°C for 10 minutes to inactivate the ligase.
4. Place the reaction on ice and proceed to Section 3.6.
3
3.6 Phosphorylation of EcoR I-Adapted cDNA
1. Add 3 µL of T4 Polynucleotide Kinase to the reaction from step 4 of Section
3.5.
2. Mix gently and incubate the reaction for 30 minutes at 37°C. Note: While the
reaction is incubating, you may begin performing steps 1 through 3 of Section
3.7.
3. Heat the reaction at 70°C for 10 minutes to inactivate the kinase.
4. Place the reaction on ice and proceed to Section 3.7.
3.7 Column Chromatography
Note: Do not add more than one reaction
per column. Addition of multiple reactions
will result in poor cloning efficiency.
Note: If the flow rate is noticeably slower
than 20 min/mL, do not use the column.
Additionally, if the drop size from the
column is not ~25 to 35 µL, do not use the
column. The integrity and resolution of the
cDNA might be compromised.
Tip: When collecting fractions, wear
gloves that have been rinsed with ethanol
to reduce static. Also, position the tube
1 to 2 cm from the bottom of the column to
avoid the effects of static on drop size.
This procedure optimizes size fractionation of the cDNA and makes the cloning of
larger inserts more probable. This procedure also ensures that residual adapters do
not enter into the library. Failure to adhere to these instructions can compromise the
quality of your cDNA library.
1. Place one of the cDNA size fractionation columns in a support. Remove the
top cap first, and then the bottom cap. Allow the excess liquid (20% ethanol) to
drain.
2. Pipet 0.8 mL of TEN buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA,
25 mM NaCl; autoclaved] onto the upper frit and let it drain completely. Repeat
this step three more times for a total of 3.2 mL. Each 0.8-mL wash will take
approximately 15 minutes, but it is important to do all four washes to remove
the 20% ethanol from the column.
3. Label 20 sterile microcentrifuge tubes from 1 to 20, and place them in a rack
with tube 1 under the outlet of the column.
4. Add 97 µL of TEN buffer to the cDNA reaction from step 4 of Section 3.6 and
mix gently.
5. Add the entire sample to the center of the top frit and let it drain into the bed.
Collect the effluent into tube 1.
6. Add 100 µL of TEN buffer to the column and collect the effluent into tube 2.
Note: Let the column drain completely—in other words, until it stops
dripping—before the addition of each new 100-µL aliquot.
7. Beginning with the next 100-µL aliquot of TEN buffer, collect single-drop
(~35 µL) fractions into individual tubes. Continue adding 100-µL aliquots of
TEN buffer until you have collected a total of 18 drops into tubes 3 through 20,
one drop per tube.
17
Methods
8.
9.
10.
11.
12.
13.
14.
15.
Using an automatic pipet, measure the volume in each tube; use a fresh tip for
each fraction to avoid cross-contamination. Record each value in column A of
one of the following work tables, using the sample table as a guide. Cap each
tube after the volume has been measured and recorded. Calculate the
cumulative elution volume with the addition of each fraction and record this
value in column B. Note: The sample table contains data typical for the size
fractionation protocol when oligo(dT) has been used to prime first-strand cDNA
synthesis; actual data will vary. The sample is provided merely to illustrate the
decision-making process for selecting cDNA for the vector ligation reaction.
Identify the fraction for which the value in column B is closest to, but not
exceeding, 600 µL (corresponding to fraction 12 in the sample table). Draw a
horizontal line across the table immediately below this fraction. Do not use any
of the subsequent fractions for your cDNA library; remove them to a separate
tube rack to avoid accidentally using them in the remainder of the protocol.
Important: Fractions collected after the 600-µL cutoff point (corresponding to
tubes 13 through 20 in the sample table) will contain smaller cDNAs and
unligated adapters. Use of these fractions significantly increases the risk of
cloning the EcoR I (Not I) adapters. In some cases with random hexamerprimed reactions, the target cDNA may elute in a later fraction, in which case
taking fractions beyond the passage of 600 µL may be a necessary risk.
Place the remaining tubes in a scintillation counter and obtain Cerenkov
counts for each fraction. Count the entire sample in the tritium channel; do not
add scintillation fluid to the tubes. In column C, record the counts
corresponding to each fraction. Note: Cerenkov counts above background
should appear after passage of 450–500 µL of buffer.
For each fraction in which the Cerenkov counts exceed background
(corresponding to fractions 8 to 12 in the sample table), calculate the amount
of cDNA, using equation 5 in Section 3.12, Analysis of cDNA from the
cDNA Size Fractionation Column. Record each cDNA amount in column D.
Divide each cDNA amount in column D by the fraction volume given in column
A to determine the cDNA concentration per fraction. Record this value in
column E.
The plasmid vector ligation reaction in Section 3.8 requires 10 ng of cDNA at
≥1 ng/µL. The λ vector ligation in Section 3.9 requires 50 ng of cDNA as a
dried pellet. Examine the data entered in columns D and E of your work table
and decide which fractions to pool and precipitate early fractions, or (if
applicable), to use the appropriate amount of cDNA from a suitable fraction
directly in the selected vector ligation reaction. Guidelines for making this
decision are provided in Section 3.12, Analysis of cDNA from the cDNA Size
Fractionation Column.
If cDNA from two or more fractions must be pooled to obtain the cDNA needed
for the vector ligation reaction, uncap the first selected tube (corresponding to
fraction 8 in the sample table), and add cDNA from each subsequent fraction
until there is the correct amount of cDNA (as determined in step 13) in the
tube. Measure the volume and add 5 µL of Yeast tRNA to the tube.
Add 0.5 volumes of NH4OAc, followed by 2 volumes of absolute ethanol
(–20°C). Vortex the mixture thoroughly and immediately centrifuge at room
temperature for 20 minutes at 14,000 × g. Note: If you are stopping here (see
“Time Planning” under Section 3.2, General Comments), store the tubes at
–20°C overnight before centrifugation to minimize the effects of 32P decay.
16. Remove the supernatant carefully and overlay the pellet with 0.5 mL of 70%
ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the
supernatant.
17. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol. To ligate
to a plasmid vector, dissolve the cDNA in 10 µL of TEN buffer and proceed to
Section 3.8. For ligation to a λ vector, proceed directly to Section 3.9.
18
3
Sample Experiment
No.
Experiment 1
A
B
C
D
Fraction
Volume
(µL)
Total
Volume
(µL)
Cerenkov
Counts
(CPM)
Amount
of cDNA
(ng)
E
Concentration of
cDNA
(ng/µL)
No.
1150 150
25
1
297 247
30
2
334 281
32
3
430 311
20
4
535 346
29
5
633 379
32
6
734 413
43
7
834 447 125 3.3 0.1
8
936 483 62516
0.44
9
1034 517 1,196 32
0.94
10
1134 551 1,740 46
1.4
11
1234 585 1,523 40
1.2
12
1334 619
13
1430 649
14
1533 682
15
1635 717
16
1732 749
17
1836 785
18
1934 819
19
2035 854
20
A
B
C
D
Fraction
Volume
(µL)
Total
Volume
(µL)
Cerenkov
Counts
(CPM)
Amount
of cDNA
(ng)
E
Concentration of
cDNA
(ng/µL)
19
Methods
Experiment 2
No.
Experiment 3
A
B
C
D
Fraction
Volume
(µL)
Total
Volume
(µL)
Cerenkov
Counts
(CPM)
Amount
of cDNA
(ng)
E
Concentration of
cDNA
(ng/µL)
No.
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
20
A
B
C
D
Fraction
Volume
(µL)
Total
Volume
(µL)
Cerenkov
Counts
(CPM)
Amount
of cDNA
(ng)
E
Concentration of
cDNA
(ng/µL)
3.8 Ligation of cDNA to a Plasmid Vector
If you intend to ligate the cDNA to a λ vector, use Section 3.9 or 3.10. This protocol
is intended for use with 10 ng of cDNA. Note: Do not proceed with this protocol until
you have made the appropriate decisions regarding the choice of fractions for use in
the ligation reaction. For more information, refer to Section 3.12, Analysis of cDNA
from the cDNA Size Fractionation Column.
Note: Although 10 to 20 ng of cDNA
generally saturates the vector, the amount
of cDNA that yields the maximal number
of clones may be higher (e.g., 40 ng). As
much as 14 µL of cDNA in TEN buffer may
be added to the ligation reaction.
1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge
tube:
ComponentAmount
5X T4 DNA Ligase Buffer.............................................................................4 µL
plasmid vector, EcoR I-cut, dephosphorylated (50 ng/µL)..........................50 ng
cDNA (≥1 ng/µL).........................................................................................10 ng
DEPC-Treated Water...............................sufficient to bring the volume to 19 µL
2. Add 1 µL of T4 DNA Ligase and mix by pipetting.
Final composition of the reaction:
50 mM Tris-HCl (pH 7.6)
10 mM MgCl2
1 mM ATP
5% (w/v) PEG 8000
1 mM DTT
2.5 µg/mL plasmid vector, EcoR I-Cut
0.5 µg/mL cDNA
50 units/mL T4 DNA ligase
3. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C.
Note: Following incubation, the cDNA will be ligated into the cloning vector and
ready for transformation into E.coli cells such as MAX Efficiency® DH5α™ or
DH10B ™ Competent Cells, or for precipitation procedures prior to
electroporation into cells such as ElectroMAX™ DH10B Cells. See Section 6
for information on these products.
3
3.9 Ligation of cDNA to λgt11 and λgt10 Vectors
If you intend to ligate the cDNA to a plasmid vector, use Section 3.8. This protocol is
intended for use with 50 ng of cDNA as a dried pellet. Note: Do not proceed with
this protocol until you have made the appropriate decisions regarding the choice of
fractions for use in the ligation reaction. For more information, refer to Section 3.12,
Analysis of cDNA from the cDNA Size Fractionation Column.
Note: Although 20 to 50 ng of cDNA
generally saturates the vector, the amount
of cDNA can be varied to determine what
quantity yields the maximal number of
clones.
1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge
tube containing the dried cDNA pellet:
ComponentAmount
5X T4 DNA Ligase Buffer..............................................................................1 µL
λ vector, EcoR I Arms (250 ng/µL).............................................................500 ng
cDNA (as a dried pellet)..............................................................................50 ng
DEPC-treated water................................... sufficient to bring the volume to 4 µL
Mix by pipetting to ensure that the cDNA is completely dissolved.
2. Add 1 µL of T4 DNA Ligase and mix by pipetting.
Final composition of the reaction:
50 mM Tris-HCl (pH 7.6)
10 mM MgCl2
1 mM ATP
5% (w/v) PEG 8000
1 mM DTT
100 µg/mL λ vector, EcoR I Arms
10 µg/mL cDNA
200 units/mL T4 DNA ligase
21
Methods
3.
Let the reaction incubate for 3 hours at room temperature or overnight at 4°C.
Note: Following incubation, the cDNA will be ligated into the cloning vector and
ready for in vitro packaging.
3.10Ligation of cDNA to a λZipLox Vector
1.
Note: Although 20 to 50 ng of cDNA
generally saturates the vector, the amount
of cDNA can be varied to determine what
quantity yields the maximal number of
clones.
Prepare a 5X DNA ligase buffer separately. Do not use the 5X T4 DNA Ligase
Buffer supplied with this system; it contains PEG which inhibits the packaging
of λZipLox. Mix 10 µL of the 10X DNA Ligase Buffer, supplied with the λZipLox
[400 mM Tris-HCl (pH 7.5), 100 mM MgCl 2, 15 mM ATP] with 10 µL of
100 mM DTT in a microcentrifuge tube on ice.
2. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge
tube containing the dried cDNA pellet:
ComponentAmount
5X DNA Ligase buffer (from step 1)...............................................................1 µL
λZipLox Arms, Not I - Sal I (250 ng/µL)........................................................ 2 µL
cDNA (as a dried pellet).....................................................................20 to 50 ng
distilled water.................................................................................................1 µL
Mix by pipetting to ensure that the cDNA is completely dissolved.
3. Add 1 µL (one unit) of T4 DNA ligase and mix gently by pipetting.
4. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C.
5. Following incubation, the cDNA will be ligated into the cloning vector and ready
for in vitro packaging.
3.11Analysis of cDNA Products
3.11.1First-Strand Yield
The overall yield of the first-strand reaction is calculated from the amount of acidprecipitable radioactivity determined as described in Section 3.3. In order to perform
the calculation, you must first determine the specific activity (SA) of the radioisotope
in the reaction. The specific activity is defined as the counts per minute (cpm) of an
aliquot of the reaction divided by the quantity (in pmol) of the same nucleotide in the
aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:
cpm/10 µL
SA (cpm/pmol dCTP) =
200 pmol dCTP/10 µL
[1]
The amount of dCTP contributed by the radiolabeled material is insignificant relative
to the unlabeled nucleotide and is ignored in equation 1.
Once the specific activity is known, the amount of cDNA in the first-strand reaction
can be calculated from the amount of acid-precipitable radioactivity determined from
the washed filter:
Amount of (cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP)
=
ds cDNA (µg)
(cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)
[2]
The correction in the numerator takes into account that, on the average, four
nucleotides will be incorporated into the cDNA for every dCTP scored by this assay.
The factor in the denominator is the amount of nucleotide that corresponds to 1 µg
of single-stranded DNA.
Example: The unwashed filter gave 50,000 cpm when it was counted. The specific
activity of the dCTP is given by equation 1:
50,000 cpm/10 µL
SA (cpm/pmol dCTP) =
200 pmol dCTP/10 µL
22
= 250 cpm/pmol dCTP
If 2 µg of starting mRNA was used and the washed filter gave 1,800 cpm, then the
amount of cDNA is calculated using equation 2:
Amount of(1,800 cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP)
=
ds cDNA (µg) (250 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)
=
0.5 µg first-strand cDNA
This amount of first-strand cDNA would represent a 25% yield relative to the 2 µg of
mRNA starting material.
3
3.11.2Second-Strand Yield
The overall yield of the second-strand reaction is calculated from the amount of
acid-precipitable radioactivity determined as described in Section 3.4. In order to
perform the calculation, you must first determine the specific activity of the
radioisotope in the reaction. The specific activity is defined as the counts per minute
of an aliquot of the reaction divided by the quantity of the same nucleotide in the
aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:
cpm/10 µL
SA (cpm/pmol dCTP)=
500 pmol dCTP/10 µL
[3]
The amount of dCTP contributed by the radiolabeled material is insignificant relative
to the unlabeled nucleotide and is ignored in equation 3.
Once the specific activity is known, the amount of cDNA in the second-strand
reaction can be calculated from the amount of acid-precipitable radioactivity
determined from the washed filter:
Amount of (cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol dCTP)
=
[4]
cDNA (µg)
(cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)
The correction in the numerator takes into account that, on the average, four
nucleotides will be incorporated into the cDNA for every dCTP scored by this assay.
The factor in the denominator is the amount of nucleotide that corresponds to 1 µg
of single-stranded DNA.
Example: The unwashed filter gave 300,000 cpm when it was counted. The specific
activity of the dCTP is given by equation 3:
300,000 cpm/10 µL
SA (cpm/pmol dCTP)=
500 pmol dCTP/10 µL
=
600 cpm/pmol dCTP
If 2 µg of starting mRNA was used and the washed filter gave 2,500 cpm, then the
amount of cDNA is calculated using equation 4:
Amount of (2,500 cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol
dCTP)
=
cDNA (µg)
(600 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)
= 0.4 µg second-strand cDNA
3.11.3Gel Analysis
The first or second-strand cDNA, if labeled with 32P, can be analyzed by alkaline
agarose gel electrophoresis to estimate the size range of products synthesized
(15,28).
The ethanol-precipitated first-strand sample is dissolved in 10 µL 1X alkaline
agarose gel sample buffer [30 mM NaOH, 1 mM EDTA, 10% (v/v) glycerol, 0.01%
bromophenol blue]. Other samples (such as the 1 Kb DNA Ladder, labeled with 32P)
can be electrophoresed after addition of a suitable volume of a more concentrated
sample buffer; the only precaution is to chelate any Mg2+ by addition of EDTA prior
to adding the alkaline sample buffer.
23
Second-Strand
The gel [1.4% (w/v)] should be cast in the appropriate volume of 30 mM NaCl,
2 mMEDTA (alkaline buffer cannot be used because it will degrade the agarose
when the solution is microwaved to melt the agarose) and should be equilibrated for
2 to 3 hours in alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA) before
loading the samples. Electrophoresis should be for 5 to 6 hours at 50 V or for 16 to
18 hours at 15 V. The gel should be dehydrated under vacuum until the buffer is
removed, then under heat and vacuum for several hours to complete the drying.
The dried gel should then be exposed to x-ray film overnight at room temperature.
First-Strand
kb
Marker
Methods
7.1
6.1
5.1
4.0
3.0
2.0
1.6
1.0
When a heterogeneous mRNA population is fractionated by alkaline gel
electrophoresis, a continuum of fragments ranging in size from 500 to 5,000
nucleotides makes up the bulk of the first and second-strand cDNA. Figure 5 shows
an alkaline electrophoretic analysis of 32P-labeled first and second-strand cDNA
synthesized from oligo(dT)-primed HeLa mRNA with the SuperScript® Choice
System.
3.12Analysis of cDNA from the cDNA Size Fractionation
Column
Calculation of the amount of size-fractionated cDNA in each column fraction is
necessary to ensure that the proper decisions are made concerning fraction
selection and that the cDNA is used economically in the ligation reaction. The
Cerenkov counts are approximately 50% of the radioactivity that would be
measured in scintillant. The counts are converted into nanograms of cDNA using the
specific activity determined in Section 3.3 or 3.4. The amount of cDNA (as double
strand) in each fraction is given by the following relationship:
Amount of (Cerenkov cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA)
=
[5]
ds cDNA (ng)
SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)
Example: If one of the fractions from the column gave 1,500 cpm when counted by
Cerenkov radiation (first-strand-labeled), then the amount of cDNA in that fraction is
calculated using equation 5:
Amount of (1,500 cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA)
=
ds cDNA (ng) SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)
0.5
Figure 5. Alkaline agarose gel analysis of first and second-strand cDNA
synthesized with the SuperScript ®
Choice System. Samples of 32P-labeled
first or second-strand cDNA made from
HeLa mRNA were ethanol-precipitated,
dissolved in alkaline agarose sample
buffer, and electrophoresed on a 1.4%
agarose gel at 15 V for 16 h.
=
32 ng
After calculating the amount and the concentration of cDNA in each fraction
(columns D and E of the work table), you are ready to select and recover cDNA for
use in the vector ligation reaction in Section 3.8, 3.9, or 3.10. Depending on which
type of vector you intend to use, certain considerations should be noted:
Recovering cDNA for ligation to plasmid vectors. If you wish to maximize the
average insert size in a plasmid-based cDNA library and your earliest selected
fraction (corresponding to fraction 8 in the sample table) contains less than the
10 ng of cDNA required for Section 3.8, you will need to pool cDNA from this fraction
with at least a portion of subsequent fractions. Because the resulting cDNA solution
will be too dilute for use in the ligation reaction, you will also need to ethanolprecipitate the pooled cDNA. For example, fraction 8 from the sample table in
Section 3.7 contains 3.3 ng of cDNA in 34 µL, and fraction 9 contains 16 ng of cDNA
in 36 µL. If all of fraction 8 is combined with 15 µL from fraction 9, the pool will
contain 10 ng of cDNA in 49 µL. The ethanol precipitation steps in Section 3.7 will
then concentrate the 10 ng cDNA in preparation for the plasmid vector ligation
reaction in Section 3.8.
If any of the selected fractions contain ≥10 ng of cDNA at ≥1 ng/µL, you can use
10 ng from the fraction directly in the plasmid vector ligation reaction in Section 3.8.
Note: If this fraction is not the earliest selected, based on Cerenkov counts
(corresponding to fraction 8 in the sample table), the average insert size in your
cDNA library will be smaller than could be obtained through additional pooling and
24
ethanol precipitation steps. Figure 6 provides an electrophoretic display of the size
ranges of cDNA obtained in fractions 8 through 19 in the experiment that generated
the sample table data.
Recovering cDNA for ligation to λ vectors. If you wish to maximize the average
insert size in a λ-based cDNA library and your earliest selected fraction
(corresponding to fraction 8 in the sample table) contains less than the 50 ng of
cDNA required for Section 3.9 or 3.10, you will need to pool cDNA from this fraction
with at least a portion of subsequent fractions. Because the cDNA for use in Section
3.9 or 3.10 must be in the form of a dry pellet, you will need to ethanol-precipitate
the cDNA, pooled or not. For example, pooling all of fractions 8, 9, and 10 from the
sample table yields ~50 ng of cDNA in solution; the ethanol precipitation steps in
Section 3.7 will then concentrate the cDNA, and 50 ng from the sample can be used
in the λ vector ligation reaction in Section 3.9 or 3.10.
3
The preceding discussion assumes that you wish to achieve maximum
transformation efficiencies in keeping with the design of Section 3.8, 3.9, or 3.10.
One other procedural option is available: if you wish merely to maximize insert size
and are willing to accept lower transformation efficiencies to attain this goal, you
may use your earliest selected fraction in Section 3.8, 3.9, or 3.10 even if it contains
less than the required amount of cDNA.
Please note that the above option should preferably be attempted in plasmid vectors
using electroporation methods, or in λ vectors using in vitro packaging methods,
since these methods offer higher cloning efficiencies (and thus a greater chance of
generating a sufficiently large cDNA library).
kb
8
9
10
11
Column Fraction
12 13
14 15
16
17
18
12.2
7.1
5.1
3.0
2.0
1.6
1.0
Figure 6. Electrophoretic analysis of size-fractionated cDNA. [32P]cDNA was fractionated
on a 1-mL prepacked column equilibrated in TEN buffer. Single-drop fractions (~35 µL each)
were collected, and aliquots were analyzed by electrophoresis on a 1% agarose gel in 40 mM
Tris-acetate (pH 8.3), 5 mM sodium acetate, 1 mM EDTA. The gel was electrophoresed at
200 V for 2 hours.
25
4
Troubleshooting
4.1 Isolation of mRNA
Note: TRIzol® Reagent can be used
to isolate high quality total RNA from
cells and tissue.
In the first step of cDNA library construction, RT converts the sequence
information of the mRNA to first-strand cDNA. The quality of the mRNA used
as the template will influence profoundly the yield and size distribution of the
first-strand product. We recommend a guanidine isothiocyanate-based
homogenization procedure to ensure rapid inactivation of RNases and
general deproteinization, followed by two selections of mRNA by oligo(dT)
cellulose chromatography to enrich maximally for poly(A)+ RNA.
The mRNA preparation can be analyzed by formaldehyde agarose gel
electrophoresis (16,29) and ethidium bromide staining (17,30). The gel
should reveal a smear of fluorescent material, and perhaps some discrete
fragments corresponding to abundant mRNAs. Residual 18S or 28S rRNAs
(approximately 2,000 or 5,000 bases in length, respectively, in mammalian
cells) will be visible even in mRNA preparations highly enriched for poly(A)+
RNA, and will be indicative of an intact mRNA population. If neither rRNA is
visible and the distribution of the mRNA is not centered in the 1- to 3-kb
range, then you will need to consider troubleshooting your RNA isolation
procedure. Examples of representative mRNA preparations have been
published (31).
4.2 First-Strand Reaction
The conditions described for the first-strand reaction have been optimized;
thus it is imperative to follow Section 3.3 explicitly. Do not increase the size
of the first-strand reaction from 20 µL.
First-strand yields will vary widely; using 2 µg of mRNA in the first-strand
reaction, we routinely obtain 25% to 35% yields from HeLa mRNA
preparations primed with oligo(dT)12-18 and 25% to 60% yields from the HeLa
mRNA preparations primed with 50 to 150 ng random hexamers, either by
themselves or in combination with oligo(dT). Analysis of the products by
alkaline gel electrophoresis reveals a distribution from 0.5 to >7 kb (see
figure 5). A lower yield does not necessarily indicate that a library cannot be
made, so long as the size distribution of the products is consistent with the
size distribution of the mRNA. A lower size distribution, if the reaction is
primed with oligo(dT), suggests RNase contamination; however, some
random hexamer-primed reactions may inherently result in a lower size
distribution due to multiple priming starts throughout the RNA template. If
RNase contamination is suspected, use the control RNA to synthesize firststrand cDNA and examine the products by alkaline gel electrophoresis.
If you obtain a poor yield in the first-strand reaction, the control RNA can be
used to verify that the system components are working properly. The control
RNA is 2.0-kb single transcript that can be substituted directly into the firststrand reaction as described in Section 3.3 [use of the extra components
provided (see Section 3.2.4, Utilization of Reagents) allows you to reverse
transcribe the control RNA without losing the capability to construct three
cDNA libraries as described in Sections 3.3 through 3.9]. The first-strand
yield using the control RNA is generally 30% to 50%. If desired, the control
RNA can be taken through the entire procedure and cloned into the vector of
choice.
26
4.3 Second-Strand Reaction
The second-strand reaction in Section 3.4 is not sensitive to RNase contamina­tion,
but strict adherence to good laboratory practice is still required. The second-strand
reaction is generally efficient, and yields of 80% to 100% (relative to the amount of
first-strand cDNA synthesized) are common. The distribution of second-strand
cDNA products should look like the distribution of the first-strand cDNA products
when analyzed by alkaline agarose gel electrophoresis (see Figure 5).
4
Unlike some second-strand procedures that do not require E. coli RNase H (13),
this enzyme must be included to provide initiation points for nick translation by DNA
polymerase I when first-strand cDNA is synthesized by SuperScript ® II RT.
Furthermore, the reaction must be incubated at ≤16°C to prevent spurious
synthesis by DNA polymerase I, although this is contrary to the original descriptions
in the literature (13,14).
Dilution of the first-strand reaction precisely as specified in Section 3.3 is extremely
important because the pH of the second-strand reaction differs from that of the firststrand reaction. This pH change influences the activity of the 3´→5´ and 5´→3´
exonucleases of DNA polymerase I, thereby limiting the number of nucleotides that
are removed from the termini of the cDNA as the second strands are completed.
4.4 EcoR I (Not I) Adapter Addition
This protocol is the most difficult to troubleshoot because it will not be suspect until
the cloning efficiency is determined. The adapter addition step is assayed most
readily by nondenaturing polyacrylamide gel electrophoresis. If a 10-µL aliquot of
the adapter ligation reaction is electrophoresed on a 12% acrylamide gel and the
DNA is visualized by ethidium bromide staining, the ligation of two adapters to each
other at their blunt ends will be evidenced by a conspicuous 38-bp fragment.
Although this does not verify that the adapters have ligated to the cDNA, it does
show that the ligation reaction in Section 3.5 is functionally sound.
4.5 Phosphorylation of EcoR I-Adapted cDNA
Problems with phosphorylation will not be apparent until the cloning efficiency is
determined. To test the phosphorylation protocol, perform a control reaction as
follows:
1.
Add the following to a sterile 1.5-mL microcentrifuge tube:
Component
Volume (µL)
5X Adapter Buffer..............................................................................................3
EcoR I (Not I) Adapters.....................................................................................1
DEPC-Treated Water.........................................................................................7
0.1 M DTT..........................................................................................................2
[γ-32P]ATP (5 µCi/µL).........................................................................................1
T4 Polynucleotide Kinase..................................................................................1
Final volume....................................................................................................15
2. Mix gently and incubate the reaction at 37°C for 30 minutes.
3. Heat the reaction at 70°C for 10 minutes to inactivate the kinase.
4. Spot duplicate 1-µL aliquots on DE-81 ion-exchange paper. Dry one filter to
determine the amount of isotope added. Wash the other filter twice in 0.3 M
ammonium formate at room temperature to remove unincorporated isotope,
rinse with water, and allow to dry.
27
Troubleshooting
5. Count both samples in standard fluor and determine the percentage of
adapters phosphorylated as follows:
% adapters phosphorylated =
cpm of washed filter
× 100
0.006 × cpm of unwashed filter
You should obtain 70% to 100% phosphorylation of the EcoR I (Not I) adapters by
this assay. Caution: If 32P-labeled adapters are substituted for unlabeled adapters
in Section 3.5, you will be unable to quantitate the amount of DNA recovered after
column chromatography, as described in Section 3.12, Analysis of cDNA from the
cDNA Size Fractionation Column. Use 32P-labeled adapters only to troubleshoot
Section 3.6.
4.6 Column Chromatography
The most likely problems with Section 3.7 will arise either from running the column
too quickly—it is imperative to let it go dry between 100-µL aliquots—or taking
fractions beyond 600 µL. If fractions beyond 600 µL are used, the library will contain
more small cDNAs (100–500 bp) or apparently “empty” clones. The empty clones
arise from ligation of the EcoR I (Not I) adapters. Please refer to Section 3.12,
Analysis of cDNA from the cDNA Size Fractionation Column and the sample table in
Section 3.7 for representative size fractionation results and for guidelines on
selecting the proper fractions.
4.7 Ligation of cDNA to a Plasmid Vector
If the transformation efficiency is low, the plasmid ligation reaction in Section 3.8
may not be proceeding properly. If the remainder of the ligation reaction is
electrophoresed on an agarose gel (along with 1 µL of the vector as a control, run in
an adjacent lane) and visualized by ethidium bromide staining (using a strong
254-nm UV transluminator), the DNA should smear upward from the position of the
vector. If the DNAs did not ligate, repeat Section 3.8 using a smaller volume (for
example, 2 µL) of the column fraction containing the highest concentration of cDNA;
however, do not go beyond the first five fractions that contain cDNA. This procedure
will minimize inhibition if the column buffer is a problem, although up to 14 µL of the
column buffer generally can be used in the ligation.
Another cause of low transformation efficiencies is using an insufficient amount of
cDNA in the ligation reaction. Recheck all calculations used to generate the data for
the table to rule out the possibility of a simple mathematical error. If the described
ligation conditions do not yield enough transformants to make your library complete,
the vector-to-cDNA ratio yielding the maximum number of clones should be
determined empirically. Also, if you chose not to ethanol-precipitate the cDNA, when
it would have been the recommended procedure following size fractionation, return
to step 13 of Section 3.7 to ethanol-precipitate the cDNA and proceed to Section
3.8.
4.8 Ligation of cDNA to a λ Vector
If the in vitro packaging efficiency is low, the λ ligation reaction in Section 3.9 or 3.10
may not be proceeding properly. If 2 µL of the ligation reaction is electrophoresed on
an agarose gel (along with 1 µL of the vector as a control, run in an adjacent lane)
and visualized by ethidium bromide staining (using a strong 254-nm UV
transluminator), the DNA should smear upward from the position of the λ vector
arms. If the DNA did not ligate, repeat Section 3.9 or 3.10.
Again, recheck all calculations to rule out the possibility of an insufficient amount of
cDNA in the ligation reaction.
28
References
5
1. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408.
2. Binns, M.M., Boursnell, M.E.G., Foulds, I.J., and Brown, T.D.K. (1985) J. Virol.
Methods 11, 265.
3. Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K.,
and Oppermann, H. (1990) EMBO J. 9, 2085.
4. Birnstiel, M.L., Busslinger, M., and Strub, K. (1985) Cell 41, 349.
5. Dowling, P.C., Giorgi, C., Roux, L., Dethlefsen, L.A., Galantowicz, M.E., Blumberg,
B.M., and Kalakofsky, D. (1983) Proc. Natl. Acad. Sci. USA 80, 5213.
6. Gruber, C.E., Cain, C., and D'Alessio, J.M. (1991) Focus® 13, 88.
7. Kotewicz, M.L., D’Alessio, J.M., Driftmeir, K.M., Blodgett, K.P., and Gerard, G.F.
(1985) Gene 35, 249.
8. Kotewicz, M.L., Sampson, C.M., D’Alessio, J.M., and Gerard, G.F. (1988) Nucl.
Acids Res. 16, 265.
9. Berger, S.L., Wallace, D.M., Puskas, R.S., and Eschenfeldt, W.H. (1983)
Biochemistry 22, 2365.
10. Gerard, G.F., D’Alessio, J.M., and Kotewicz, M.L. (1989) Focus 11, 66.
11. D’Alessio, J.M., Gruber, C.E., Cain, C., and Noon, M.C. (1990) Focus 12, 47.
12. Gerard, G.F., Schmit, B.J., Kotewicz, M.L., and Campbell, J.H. (1992) Focus 14,
91.
13. Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161.
14. Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263.
15. D’Alessio, J.M. and Gerard, G.F. (1988) Nucl. Acids Res. 16, 1999.
16. Huynh, T.V., Young, R.A., and Davis, R.W. (1985) in DNA Cloning: A Practical
Approach (Vol. 1) (D.M. Glover, ed.), IRL Press Limited, Oxford, England,
p. 49.
17. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.
18. Young, R.A. and Davis, R.W. (1983) Science 222, 778.
19. Helfman, D.M., Feramisco, J.R., Fiddes, J.C., Thomas, G.P., and Hughes, S.H.
(1983) Proc. Natl. Acad. Sci. USA 80, 31.
20. Grunstein, M. and Hogness, D. (1975) Proc. Natl. Acad. Sci. USA 72, 3961.
21. Benton, W.D. and Davis, R.W. (1977) Science 196, 180.
22. Vieira, J. and Messing, J. (1987) Methods Enzymol. 153, 3.
23. Huang, M.T.F. and Gorman, C.M. (1990) Mol. Cell. Biol. 10, 1805.
24. Murray, N.E., Brammar, W.J., and Murray, K. (1977) Mol. & Gen. Genet. 150, 53.
25. Mierendorf, R.C., Percy, C., and Young, R.A. (1987) Methods Enzymol. 152, 458.
26. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
27. Simms, D. (1995) Focus 17, 39.
28. McDonnell, M.W., Simon, M.N., and Studier, F.W. (1977) J. Mol. Biol. 110, 119.
29. Gerard, G.F. and Miller, K. (1986) Focus 8:3, 5.
30. Matathias, A.S. and Komro, C. (1989) Focus 11, 79.
31. D’Alessio, J.M. and Noon, M.C. (1989) Focus 11, 49.
29
6
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18 January 2013