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For life science research only. Not for use in
diagnostic procedures.
SP6/T7 Transcription Kit
For in vitro transcription of DNA
Cat. No. 10 999 644 001
Kit for 2 × 20 standard transcription assays
y Version 18
Content version: August 2015
Store at ⫺15 to ⫺25° C
1. Kit Contents
Bottle/
Cap
Figure 1
Label
Content and function
1
pSPT18 DNA
• 40 µl pSPT18 DNA, 0.25 mg/ml
2
pSPT19 DNA
• 40 µl pSPT19 DNA, 0.25 mg/ml
3
Control DNA
• 20 µl DNA mixture
• 5 µg each of pSPT18- and pSPT19-neo-DNA,
cleaved with Eco RI
4
ATP
• 40 µl, 10 mM ATP in Tris buffer
5
CTP
• 40 µl, 10 mM CTP in Tris buffer
6
GTP
• 40 µl,10 mM GTP in Tris buffer
7
UTP
• 40 µl, 10 mM UTP in Tris buffer
8
10 × Buffer
• 100 µl
• 10x concentrated transcription buffer
9
DNase I, RNase- • 20 µl DNase I, RNase-free
free
• [10 U/µl] in buffer with 50% glycerol
10
RNase inhibitor • 20 µl RNase inhibitor
• [20 U/µl] in buffer with 50% glycerol
11
SP6 RNA polymerase
• 20 µl SP6 RNA polymerase
• [10 U/µl] in buffer with 50% glycerol
12
T7 RNA polymerase
• 20 µl T7 RNA polymerase
• [10 U/µl] in buffer with 50% glycerol
Storage and
Stability
Nhe l
2863
Nae l
2832
Eco 47lll
2739
The unopened kit is stable at -15 to -25°C until the
expiration date printed on the label.
Note: Repeated freezing and thawing should be
avoided.
2. Product Overview
Description
DNA is inserted into the polylinker site of the transcription vectors pSPT18 or pSPT19 (see fig.); these two
vectors differ only in the orientation of their polylinker
regions. The promoters for SP6 and T7 RNA polymerases are located on either side of the polylinker.
SP6 and T7 RNA polymerases specifically transcribe
DNA sequences downstream of the SP6 or T7 promoters respectively (1, 2). Cloned inserts within the polylinker region are transcribed from either promoter. The
first DNA strand may be transcribed with SP6 RNA
polymerase and the opposite strand using T7 RNA
polymerase. It is also possible to transcribe the first and
opposite strands by inserting the same DNA into both
pSPT18 and pSPT19 in opposite orientations and
transcribing with only one of the RNA polymerases.
SP6 and T7 RNA polymerase use the cloned DNA as
template and synthesize complementary RNA in the
presence of Mg2+ and ribonucleoside triphosphates.
Spermidine stimulates enzyme activity. Specifically
labeled transcripts are obtained when using radioactively (e.g. 32P, 3H, 35S) or non radioactively (e.g.
digoxigenin or biotin) labeled ribonucleotide triphosphates.
Application
Labeled transcripts lend themselves to all DNA and
RNA hybridization techniques (3) and are also used for
genomic sequencing (4) and S1 nuclease studies (5).
Large amounts of highly pure RNA can be synthesized
using the SP6/T7 system. These transcripts are used
for studies on RNA-processing systems (6).
Synthesized RNA can be translated in vitro, or in vivo
after injection into oocytes. The transcription of defined
mRNA can be inhibited by the introduction of “antisense“-RNA (7).
The efficiency of in vivo translation of synthesized
mRNA can be increased significantly by the introduction of a cap-structure.
Sample Material
DNA inserted into the transcription vectors pSPT18 or
pSPT19.
The template DNA must be linearized with a suitable
restriction enzyme before the transcription reaction to
obtain transcripts of a defined length. Using intact
plasmid DNA as template for transcription will result in
heterogeneous transcripts of multiple plasmid lengths.
*available from Roche Diagnostics
0815.11000420001 8
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Number of
2 × 20 standard transcription assays may be performed
Labeling Reactions with the kit. However, twice as many reactions may be
performed with our control DNA (vial 3) or with highly
purified DNA using 5 units of the respective RNA polymerase per assay. This may result in approximately 3–
5% lower incorporation. Increasing the temperature to
+40°C will result in a slight increase in incorporation.
RNA may be synthesized in the presence of labeled or
unlabeled ribonucleoside triphosphates.
Advantages
Procedure
Step
1
Transcripts produced with SP6 or T7 RNA polymerases
exhibit several advantages:
• They have high levels of specific labeling.
• The length of transcripts can be exactly controlled
by linearization of the DNA template, enabling the
production of long labeled fragments (>4 kb).
• The single-stranded nature of the transcripts prevents renaturation as occurs with double-stranded
DNA probes.
• Single-stranded probes hybridize specifically only to
their complementary strand, and are, therefore, useful for screening M13 clones of specific orientation.
• The properties of DNA/RNA hybrids allow hybridization under more stringent conditions than with
labeled DNA probes.
Action
• Add the following to an microfuge tube on ice:
Reagent
0.5 ␮g DNA
Volume
Control
× ␮l
2 ␮l controlDNA (vial 3)
ATP, GTP, UTP mixture
3 ␮l
3 ␮l
10 × buffer (vial 8)
2 ␮l
2 ␮l
[␣-32P] CTP, 400 Ci/mmol,
aqueous solution
5 ␮l
5 ␮l
add double dist. water to a final
volume of 18 ␮l
× ␮l
7 ␮l
SP6 or T7 polymerase (vial 11 or
12)
1 ␮l
1 ␮l
20 ␮l
20 ␮l
Final volume
• Mix and centrifuge briefly.
• Incubate for 20 min at 37°C.
2
3. How to Use this Product
(optional)
3.1 Before You Begin
The yield and extent of specific labeling and the kinetKinetics of the
Labeling Reaction ics of the labeling reaction depend on the ratio of DNA
to labeled ribonucleoside triphosphate. The standard
assay described uses 0.5 ␮g of the corresponding template contained in the control DNA (solution 3; 0.5 ␮g
pSPT18-neo DNA when using SP6 RNA polymerase
and 0.5 ␮g pSPT19-neo DNA when using T7-RNA
polymerase, respectively) and 50 ␮Ci [␣-32P] CTP, 400
Ci/mmol. Ribonucleoside triphosphates with specific
activities of 3,000 Ci/mmol must be supplemented with
the analogous “cold“ ribonucleoside triphosphate to
obtain full length transcripts. For example, when using
[␣-32P] CTP, 3,000 Ci/mmol, supplement the standard
assay with 1 ␮l CTP (vial 5, diluted 1 + 49 with sterile
double dist. water).
• Add 2 ␮l DNase I, RNase-free (vial 9) to remove template
DNA.
• Incubate for 15 min at +37°C.
Note: Essential for RNase-protection experiments!
3
Stop the reaction by adding 2 ␮l 0.2 M EDTA (pH 8.0) and/or heating to +65°C for 10 min.
Note: The RNA transcripts can be checked for length and integrity
by native or denaturing gel electrophoresis.
4
For the removal of non-incorporated ribonucleoside triphosphates
we recommened to use:
• Quick Spin Column, Sephadex G-50 (Fine)* or
• repeated ethanol precipitation.
Determination of The level of labeling may be determined by measuring
Labeling Efficiency the radioactivity incorporated into an aliquot of the
assay in comparison to total input radioactivity. The
kinetics of incorporation may be monitored by removal
of aliquots at varying time points and trichloroacetic
acid precipitation.
Elevating the ratio of radioactive ribonucleoside triphosphate to DNA substrate, allows higher specific
labeling with retarded kinetics of reaction. Thus, using
0.5 μg of the corresponding template contained in the
control DNA and 200 μCi >40% of the input radioactivity are incorporated in 45 min.
Transcript Length Transcripts of 1028 or 1035 bases respectively are
obtained with SP6 or T7 RNA polymerase from the
of the Controls
control DNA (vial 3) in the standard assay. Under standard assay conditions >50% of the label is incorporated in 20 min into RNA transcripts.
3.3 Transcription Assay with Digoxigenin-11-UTP
3.2 Standard Labeling Assay
Additional Equip- • Digoxigenin-11-UTP*
ment and Reagents • 0.2 M EDTA (pH 8.0)
Required
• waterbath or heating block
• template DNA
Note: Template DNA must be linearized with a suitable
restriction enzyme before the transcription reaction,
using intact plasmid DNA as template for transcription
will result in heterogeneous transcripts of multiple
plasmid lengths. The DNA template may be removed
by digestion with DNase I, RNase-free, after the reaction.
Additional Equip- • 0.2 M EDTA (pH 8.0)
ment and Reagents • waterbath or heating block
Required
• template DNA
Note: Template DNA must be linearized with a suitable
restriction enzyme before the transcription reaction,
using intact plasmid DNA as template for transcription
will result in heterogeneous transcripts of multiple
plasmid lengths. The DNA template may be removed
by digestion with DNase I, RNase-free*, after the reaction.
ATP, GTP, UTP
mixture
In the following table please find a protocol for the
standard labeling reaction.
Note: 1 µl Ribonuclease inhibitor may be added to the
transcription reactions to ensure the preservation of
the full-length single-stranded RNA molecules, if considered necessary
Prepare the ATP, GTP, UTP mixture by making a 1:1:1
mixture of solution 4, solution 6, and solution 7.
Please refer to the following table:
Preparation of
.
Additional
Solutions Required
Solution
Composition
2
ATP, GTP, CTP
Mix
Mix 1:1:1 of ATP (vial 4), CTP (vial 5),
and GTP (vial 6).
UTP/DIG-11UTP Mix
Mix 1:1 Digoxigenin-11-UTP (6 mM)
with UTP (vial 7).
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Procedure
In the following table please find a protocol for the
labeling assay with Digoxigenin-11-UTP.
Note: Biotin or Fluorescein-labeled UTP can be used
in the same way.
Step
1
Procedure
Step
Action
1
• Add the following to an microfuge tube on ice:
Reagent
Volume
Control
x ␮l
4 ␮l controlDNA (vial 3)
ATP, GTP, CTP mix
6 ␮l
6 ␮l
ATP, GTP, CTP, UTP mixture
8 ␮l
8 ␮l
UTP/DIG-11-UTP mix
2 ␮l
2 ␮l
10 × buffer (vial 8)
2 ␮l
2 ␮l
10 × buffer (vial 8)
2 ␮l
2 ␮l
4 ␮l
1 µl
1 µl
add sterile double dist. water to
a final volume of 18 ␮l
× ␮l
RNase Inhibitor (vial 10)
add sterile double dist. water to
a final volume of 18 ␮l
× ␮l
3 ␮l
SP6 or T7 polymerase (vial 11 or
12)
2 ␮l
2 ␮l
SP6 or T7 polymerase (vial 11 or
12)
2 ␮l
2 ␮l
Final volume
20 ␮l
20 ␮l
20 ␮l
20 ␮l
Reagent
1 ␮g DNA
3
2
(optional)
• Add 2 ␮l DNase I, RNase-free (vial 9) to remove template
DNA.
• Incubate for 15 min at +37°C.
Note: Essential for RNase-protection experiments!
Stop the reaction by adding 2 ␮l 0.2 M EDTA (pH 8.0) and/or heating to 65°C for 10 min.
Note: The RNA transcripts can be checked for length and integrity
by native or denaturing gel electrophoresis.
Control
× ␮l
4 ␮l controlDNA (vial 3)
• Add 2 ␮l DNase I, RNase-free (vial 9) to remove template
DNA.
• Incubate for 15 min at +37°C.
Note: Essential for RNase-protection experiments!
3
Stop the reaction by adding 2 ␮l 0.2 M EDTA (pH 8.0) and/or heating to 65°C for 10 min.
Note: The RNA transcripts can be checked for length and integrity
by native or denaturing gel electrophoresis.
4
Transcripts can be purified from enzyme activity by phenol
(optional) extraction.
4. Additional Information on this Product
Determination of The labeling efficiency is determined by semi-quantitaLabeling Efficiency tive determination. The protocol is available in several
DIG Labeling kits e.g. DIG RNA Labeling Kit (SP6/T7)*
or the DIG Applications Manual for Filter Hybridization.
References
1
2
3
4
5
6
7
3.4 “Cold” Transcription
General
Volume
• Mix and centrifuge briefly.
• Incubate for 60 min at +37°C.
• Mix gently and centrifuge briefly.
• Incubate for 2 h at +37°C.
Note: Longer incubations do not increase the yield of labeled
RNA.
2
Action
• Add the following to a microfuge tube on ice:
1 ␮g DNA
Final volume
(optional)
Standard assay.
Note: The template DNA is removed by treatment with
DNase I, RNase-free. RNase inhibitor may be added to
the reaction, if considered necessary.
All four ribonucleoside triphosphates are used unlabeled for “cold” transcriptions. During “cold” transcription reactions, substrate levels are not limiting and the
synthesis continues longer producing maximum
amounts of RNA in the standard assay of 1 h
described here. Up to 15 ␮g of RNA transcripts may be
obtained from 1 ␮g of the corresponding template
contained in the control DNA.
Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477.
Kassavetis, G. A. et al. (1982), J. Biol. Chem. 257, 5779.
Southern, E. M. (1975) J. Mol. Biol. 98, 503.
Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 82, 1991.
Gannon, F. et al. (1980) Nucleic Acids Res. 8, 4405.
Peebles, C. L. et al. (1986) Cell 44, 213.
Melton, D. A. (1985) Proc. Natl. Acad. Sci. USA 82, 144.
Quality Control
• 0.2 M EDTA (pH 8.0)
Additional
Equipment and
• waterbath or heating block
Reagents Required
• template DNA
Note: Template DNA must be linearized with a suitable
restriction enzyme before the transcription reaction,
using intact plasmid DNA as template for transcription
will result in heterogeneous transcripts of multiple
plasmid lengths. The DNA template may be removed
by digestion with DNase I, RNase-free, after the reaction.
The standard assay with 1.0 µg of the corresponding
template contained in the control DNA labeled with
6 mM DIG-11-UTP mix gives sensitivity by direct spottest a 1 pg incorporation in 20 min. The transcripts
obtained are checked by gelelectrophoresis for homogeneity and full length.
ATP, GTP, CTP, UTP mixture: Prepare this mix by comAdditional
Solution Required bining solutions in vials 4, 5, 6 and 7 at a ratio of
1+1+1+1.
3
sigma-aldrich.com
Ordering Information
Kits
Product
Pack size
Cat. No.
DIG RNA Labeling
Kit (SP6/T7)
2 × 10 labeling
reactions
11 175 025 910
DIG RNA Labeling 40 ␮l (20 reacti- 11 277 073 910
ons)
Mix
Single Reagents
Product
Pack size
Cat. No.
40 ␮mol
(400 ␮l)
11 140 965 001
250 nmol (25
␮l)
11 388 908 910
40 ␮mol
(400 ␮l)
11 140 922 001
DIG-11-UTP
250 nmol (25
␮l)
11 209 256 910
Fluorescein-12UTP
250 nmol (25
␮l)
11 427 857 910
GTP, lithium salt
40 ␮mol
(400 ␮l)
11 140 957 001
NBT/BCIP Stock
Solution
8 ml
11 681 451 001
Quick Spin
Columns, Sephadex G-50
20 columns
11 273 965 001
Protector RNase
Inhibitor
2,000 U
10,000 U
[(5x 2,000 U)]
03 335 399 001
03 335 402 001
Set of ATP,CTP,
GTP, UTP Ribonucleoside triphosphate Set
1 Set
4x 20 ␮mol
11 277 057 001
SP6 RNA Polymerase
1000 units
10 810 274 001
T3 RNA Polymerase
1000 units
11 031 163 001
T7 RNA Polymerase
1000 units
10 881 767 001
UTP, lithium salt
40 ␮mol
(400 ␮l)
11 140 949 001
ATP, lithium salt
Biotin-16-UTP
CTP, lithium salt
Changes to Previ- Update of Quality Control Test
ous Version
Distributed by Sigma-Aldrich®
Editorial Changes
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respective holders.
Regulatory
Disclaimer
For life science research only. Not for use in diagnostic
procedures.
Disclaimer of
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For patent license limitations for individual products
please refer to: List of biochemical reagent products
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