In Vitro Transcription
and RNA Research
Contents
T7, T3, and SP6 RNA Polymerases.................................................. 2
E. coli RNA Polymerases (Core and Holoenzyme).................... 2
T7 R&DNA™ Polymerase.................................................................... 3
NTPs.......................................................................................................... 3
AmpliScribe™ T7-Flash™ Transcription Kit.................................. 4
AmpliScribe™ T7, T3, and SP6 High Yield
Transcription Kits.............................................................................. 4
AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit........... 5
AmpliScribe™ T7 Aminoallyl-RNA Transcription Kit................ 5
AmpliCap™ High Yield Message Maker Kits............................... 6
Cap Analogs.......................................................................................... 6
Poly(A) Polymerase Tailing Kit......................................................... 7
DuraScribe® T7 & SP6 Transcription Kits...................................... 8
ScriptGuard™ RNase Inhibitor......................................................... 9
Thermostable RNA Ligase................................................................ 9
Tobacco Acid Pyrophosphatase...................................................10
RNA 5’ Polyphosphatase.................................................................10
RiboShredder™ RNase Blend.........................................................11
E. coli RNase H.....................................................................................11
Terminator™ 5’-Phosphate-Dependent Exonuclease..........12
MonsterScript™ Reverse Transcriptase......................................13
MonsterScript™ 1st-Strand cDNA Synthesis Kit.....................13
CircLigase™ II ssDNA Ligase...........................................................14
RNA Amplification Kits....................................................................15
RNA Polymerases
T7, T3, and SP6 RNA Polymerases
The best value in phage RNA polymerases.
• High purity, promoter specificity, and activity.
• Free of detectable DNase and RNase activities.
• Function-tested in in vitro transcription reactions.
All RNA Polymerases are available in bulk. T7 and T3 RNA Polymerases are also available at
concentrations up to 2,500 Units/µl. Please contact EPICENTRE for custom orders.
Cat. # Concentration
Quantity T7 RNA Polymerase
T7905K
50 U/µl
5,000 Units
T7950K
50 U/µl
50,000 Units
TM910K
200 U/µl
10,000 Units
(Enzyme only. Transcription Buffer not included.)
T3 RNA Polymerase
T4905K
T9050K
50 U/µl
50 U/µl
5,000 Units
50,000 Units
(Enzyme only. Transcription Buffer not included.)
SP6 RNA Polymerase
S4905K
50 U/µl
5,000 Units
S4950K
50 U/µl
50,000 Units
SM910K
200 U/µl
10,000 Units
(Enzyme only. Transcription Buffer not included.)
Transcription Buffer Package
BP1001
1 Pkg
Includes 5 ml of 5X Transcription Buffer and 2.5 ml of 100 mM DTT.
E. coli RNA Polymerases (Core and Holoenzyme)
EPICENTRE is the only commercial source for both:
• Purified Core Enzyme that has no detectable sigma subunit by gel electrophoresis.
• Purified Holoenzyme that is 100% sigma-saturated for efficient transcription of DNA templates
containing promoters.
Perform rapid, high throughput screening of bacterial RNA polymerases and inhibitors using the
Kool™ NC-45 RNAP Activity and Inhibitor Screening Kit. Visit www.EpiBio.com for details.
Figure 1. Subunit patterns of E. coli
RNA Polymerase Core Enzyme and
Holoenzyme preparations. Equivalent
amounts of each enzyme were
separated by electrophoresis on an
SDS 15% polyacrylamide gel, stained
with Coomassie® Blue, and dried. The
sigma-70 subunit is 100% saturating
in the Holoenzyme, but is below
detectable limits in the Core Enzyme.
Cat. #
Quantity E. coli RNA Polymerase Core Enzyme
C90100
C90500
100 Units
500 Units
Sigma-Saturated E. coli RNA Polymerase Holoenzyme
S90050
50 Units
S90250
250 Units
2
Holo Core
– β′, β
–σ
–α
–ω
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RNA Polymerases and NTPs
T7 R&DNA™ Polymerase
T7 R&DNA™ Polymerase is a mutant form of the wild-type T7 RNA Polymerase that:
• Readily incorporates 2′-dNTPs and other 2′-modified-dNTPs, as well as canonical NTPs.
• Produces “RNA” of mixed rNMP/2′-dNMP or rNMP/2′-modified-NMP composition.
• Produces modified “RNA” transcripts that are resistant to RNase A and related RNases.
• Uses the same transcription promoters as the wild-type T7 RNA polymerases.
The DuraScribe® T7 Transcription Kit for making 2′-fluorine-modified RNA that is completely
resistant to RNase A is also available (see p. 8).
Cat. #
Concentration
Quantity T7 R&DNA™ Polymerase
D7P9201K
50 U/µl
D7P9205K
50 U/µl
Includes 5X Reaction Buffer, DTT.
1,000 Units
5,000 Units
NTPs
EPICENTRE’s NTPs are supplied as sterile, neutral aqueous solutions.
Cat. #
Concentration
Quantity ATP
R109AT
RA02825
10 mM
100 mM
5 µmol 25 µmol CTP
R109CT
RC02825
10 mM
100 mM
5 µmol 25 µmol GTP
R109GT
RG02825
10 mM
100 mM
5 µmol 25 µmol UTP
R109UT
RU02825
10 mM
100 mM
5 µmol 25 µmol One Premixed Solution of all 4 NTPs
R344NT
2.5 mM each
5 µmol each (Premix in one tube contains ATP, CTP, GTP & UTP)
One Tube of each NTP (ATP, CTP, GTP, UTP)
RN02825
100 mM each
25 µmol each
(Not premixed)
[email protected]
2′-Fluorine-dCTP (2′-F-dCTP)
R2F110C
50 mM
1 µmol
2′-Fluorine-dUTP (2′-F-dUTP) R2F110U
50 mM
1 µmol
Aminoallyl-UTP
AAU5202
50 mM 2.5 µmol
Biotin-16-UTP
BU6105H
50 mM
500 nmol
3
In Vitro Transcription Kits
AmpliScribe™ T7-Flash™ Transcription Kit
AmpliScribe™ T7-Flash™ Transcription Kit reactions:
• Are rapid (30-minute) reactions that produce more RNA than other kits produce in 2 hours.
• Transcribe 180 µg of RNA from 1 µg of DNA template.
• Produce high yields of RNA from both long and short DNA templates.
• Generate high yields of RNA even from limiting amounts of DNA template.
Table 1. An AmpliScribe™ T7-Flash™ reaction produces high yields of RNA from both long and
short DNA templates, including linearized plasmids, PCR products, and oligodeoxynucleotides.
RNA Transcript Size
26 bases
96 bases
335 bases
1.4 kb
6 kb
RNA Yield (µg)
12
36
76
176
187
Figure 1. An AmpliScribe™ T7-Flash™
transcription reaction is complete in
30 minutes and yields more RNA than
other 2-hour "high-yield" transcription
reactions. Yields shown were obtained
using 1 µg of a linear DNA template in a
20-µl reaction, producing a 1.4-kb RNA.
AmpliScribe™ T7-Flash™ Kit
Another High-Yield Kit
200
150
100
50
0
c910411
RNA Yield (µg)
RNA Yield (pmol)
1,319
1,071
648
359
89
30
60
90
120
Minutes
Cat. #
Quantity AmpliScribe™ T7-Flash™ Transcription Kit
ASF3257
25 Reactions
ASF3507
50 Reactions
Contents: AmpliScribe™ T7-Flash™ Enzyme Solution (with added RNase
Inhibitor), AmpliScribe™ T7-Flash™ 10X Reaction Buffer, 100-mM ATP,
CTP, GTP and UTP Solutions, RNase-Free Water, RNase-Free DNase I,
DTT, Control DNA Template (linearized).
AmpliScribe™ T7, T3, and SP6 High Yield Transcription Kits
• Produce up to 150 µg of RNA in a 2-hour reaction.
Cat. #
Quantity AmpliScribe™ T7 High Yield Transcription Kit
AS2607
25 Reactions
AS3107
50 Reactions
AmpliScribe™ T3 High Yield Transcription Kit
AS3103
50 Reactions
AmpliScribe™ SP6 High Yield Transcription Kit
AS3106
50 Reactions
Contents: AmpliScribe™ T7, T3, or SP6 Enzyme Solution (with added
RNase inhibitor), 100-mM ATP, CTP, GTP, and UTP Solutions,
AmpliScribe™ 10X Reaction Buffer, RNase-Free Water, RNase-Free
DNase I, DTT, Control Template DNA (linearized).
4
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In Vitro Transcription Kits
AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit
The AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit enables high-yield synthesis of biotin-RNA
or biotin-antisense RNA (aRNA, also called cRNA) for use as microarray target or as probe for
in situ or blotting experiments.
• The kit uses EPICENTRE’s AmpliScribe T7-Flash rapid, high-yield in vitro transcription technology
to generate the highest possible yield of biotin-RNA.
• Reaction conditions are optimized to yield uniformly labeled biotin-RNA for high signal intensity.
Table 1. Yield of Biotin-RNA from varying amounts of a 1.3-kb control template
DNA from an AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit reaction.
Input DNA Template
Biotin-RNA Yield (1-hour Reaction)
Biotin-RNA Yield (4-hour Reaction)
50 ng
15-25 µg
60-70 µg
500 ng
140-150 µg
>180 µg
>180 µg
>180 µg
1 µg
Cat. #
Quantity AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit
ASB71110
10 Reactions
Contents: AmpliScribe™ T7-Flash™ Enzyme Solution, ScriptGuard™
RNase Inhibitor, AmpliScribe™ T7-Flash™ 10X Reaction Buffer, NTP/
Biotin-UTP PreMix (ATP, CTP, GTP, UTP, and Biotin-16-UTP)
Solution, RNase-Free Water, RNase-Free DNase I, DTT, Control DNA
Template (linearized).
AmpliScribe™ T7 Aminoallyl-RNA Transcription Kit
The AmpliScribe™ T7 Aminoallyl-RNA Transcription Kit incorporates Aminoallyl-UTP into RNA
transcripts to generate high yields of aminoallyl-RNA (AA-RNA). The AA-RNA produced can be
subsequently labeled and used as microarray target or as probe for in situ hybridization or blotting
experiments.
200
50 ng
100 ng
500 ng
1 µg
150
100
50
0
30 min
1 hr
c110501
RNA Yield (µg)
• High yields of Aminoallyl-RNA (over 150 µg of RNA from 1 µg of DNA template).
• Reaction conditions are optimized to yield uniformly labeled aminoallyl-RNA for high signal
intensity following dye or biotin conjugation.
• Less expensive than direct incorporation of fluorescent- or biotin-labeled NTP.
Table 1. The AmpliScribe™ T7 AminoallylRNA Transcription Kit produces high yields
of aminoallyl-RNA even from limiting
amounts of DNA template.
2 hr
Reaction Time
Cat. #
Concentration
Quantity AmpliScribe™ T7 Aminoallyl-RNA Transcription Kit
AA50125
25 Reactions
Contents: AmpliScribe™ T7 Aminoallyl-RNA Enzyme Solution (with
added RNase Inhibitor), AmpliScribe™ T7 Aminoallyl-RNA 10X
Reaction Buffer, ATP, CTP, GTP, UTP and Aminoallyl-UTP Solutions,
RNase-Free Water, RNase-Free DNase I, DTT, Control DNA Template
(linearized).
Biotin-X-X-NHS
BXX51005
BXX51010
[email protected]
2.5 mg/Vial
2.5 mg/Vial
5 Vials
10 Vials
5
In Vitro Transcription Kits
AmpliCap™ High Yield Message Maker Kits
The AmpliCap-Max™ and AmpliCap™ Kits are formulated to produce exceptionally high yields of
5′-capped RNA for use in in vitro translation, microinjection, and mRNA processing studies.
• Kits incorporate m7GpppG cap analog at the 5′ end of the transcribed RNA.
• AmpliCap-Max™ T7 & T3 Kit reactions yield up to 60 µg of RNA from 1 µg of template.
• Capping efficiencies of up to 80%.
• Optimized m7GpppG/NTP PreMix is provided.
• AmpliCap-Max™ T7 and T3 Kit reactions are complete in 30 minutes.
RNA Yield (µg)
Figure 1. Thirty-minute AmpliCapMax™ T7 and T3 reactions each yield
~60 µg of 5’-capped RNA using the
respective control templates.
AmpliCap-Max™ T7
Cat. #
AmpliCap-Max™ T3
Quantity AmpliCap-Max™ T7 High Yield Message Maker Kit
ACM04037
25 Reactions
AmpliCap-Max™ T3 High Yield Message Maker Kit
ACM04033
25 Reactions
Contents: AmpliCap-Max™ T7 or T3 Enzyme Solution (with added
RNase Inhibitor), AmpliCap-Max™ Cap/NTP PreMix, 20 mM GTP,
AmpliCap-Max™ 10X Transcription Buffer, RNase-Free Water, RNaseFree DNase I, DTT, Control Template DNA (linearized).
AmpliCap™ SP6 High Yield Message Maker Kit
AC0706
25 Reactions
Contents: AmpliCap™ SP6 Enzyme Solution (with added RNase
Inhibitor), AmpliCap™ 10X Transcription Buffer, AmpliCap™ Cap/NTP
PreMix, 20 mM GTP, RNase-Free DNase I, DTT, RNase-Free Water,
Control Template DNA (linearized).
Cap Analogs
Each Cap Analog is:
• Function tested as a substrate for capping an RNA transcript synthesized in an in vitro transcription
reaction.
• Provided as a 20-mM solution.
Cat. #
Quantity Standard Cap Analog, m G[5′]ppp[5′]G Solution
C61025
1,250 nmol
7
Unmethylated Cap Analog, G[5′]ppp[5′]G Solution
C32010
500 nmol Trimethylated Cap Analog, m32,2,7G[5′]ppp[5′]G Solution
C06005
250 nmol 6
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In Vitro Transcription Kits
Poly(A) Polymerase Tailing Kit
EPICENTRE’s Poly(A) Polymerase is a cloned enzyme that uses ATP for template-independent
addition of adenosine nucleotides to the 3′-hydroxyl terminus of RNA molecules.
5’ N—OH 3’ + ATP ➞ 5’ N—AAAA(n) 3’
RNA
Poly(A) Polymerase
3’-poly(A) tailed RNA
Add a 3′-poly(A) tail to your RNA to:
• Increase the stability and enhance the translation of an RNA.
• Provide an oligo(dT) priming site to poly(A)− RNAs, such as miRNA or bacterial RNAs.
• 3′-end label RNA or miRNA.
Poly(A) tailing (Minutes)
RNA
kb MW
0′
10′
30′
60′
3.0
2.5
2.0
1.5
1.0
Figure 1. Poly(A) tailing of RNA with the Poly(A) Polymerase
Tailing Kit. The entire final product of an AmpliCap-Max™ in vitro
transcription capping reaction (60 µg of a 1,760-base luciferase
RNA) was treated with 8 units of Poly(A) Polymerase for the
indicated times. For each sample, 0.1-µg aliquots were analyzed
on a 1% agarose-formaldehyde gel.
Cat. #
Quantity Poly(A) Polymerase Tailing Kit
PAP5104H
50 Reactions (400 Units)
Contents: Poly(A) Polymerase, 10X Reaction Buffer, 10 mM ATP,
Sterile RNase-Free Water.
[email protected]
7
In Vitro Transcription Kits
DuraScribe® T7 & SP6 Transcription Kits
The DuraScribe® T7 and SP6 Transcription Kits* produce 2′-Fluorine-modified RNA transcripts—
called DuraScript® RNA—that are completely resistant to RNase A. DuraScribe T7 and SP6
RNA Polymerases are mutant forms of T7 and SP6 RNA Polymerases that efficiently incorporate
2′-Fluorine-dCTP (2′-F-dCTP) and 2′-Fluorine-dUTP (2′-F-dUTP), as well as ATP and GTP into
full-length transcripts. They use the same promoters as wild-type T7 and SP6 RNA polymerases.
The presence of the 2′-F-dC and 2′-F-dU nucleotides render DuraScript RNA completely resistant
to RNase A and related ribonucleases. A standard DuraScribe T7 reaction produces about 50 µg of
DuraScript RNA.
Use DuraScript RNA for:
• RNAi, ribozyme, and antisense RNA studies.
• In situ hybridization.
• Ribonuclease protection assays.
DuraScript® RNA Yield (µg)
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
80
Figure 1. Yield of RNA from a
DuraScribe® T7 Transcription
reaction. A standard reaction
(4-6 hours) produced 40-60 µg
of a 1.4-kb DuraScript® RNA.
60
40
20
0
2 hours
4 hours
6 hours
Incubation Time
M
1
2
3
4
Figure 2. DuraScript® RNA is resistant
to RNase A digestion. A 1.4-kb standard
RNA transcript and a 1.4-kb DuraScript
RNA transcript were each incubated
with 1 U of highly purified RNase A for
30 minutes. The standard RNA transcript
was completely degraded while the
DuraScript RNA transcript remained
intact. Lane M, Size ladder; lane 1,
1.4-kb standard RNA transcript; lane 2,
standard RNA after RNase A treatment;
lane 3, 1.4-kb DuraScript RNA; lane 4,
DuraScript RNA after RNase A treatment.
Cat. #
Quantity DuraScribe® T7 Transcription Kit
DS010910
DS010925
10 Reactions
25 Reactions
DuraScribe® SP6 Transcription Kit
DS041010
10 Reactions
Contents: DuraScribe® Enzyme Mix, DuraScribe® 10X Reaction Buffer,
ATP, GTP, 2′-F-dCTP, 2′-F-dUTP, DNase I, DTT, Control Template
DNA (linearized), RNase-Free Water.
8
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RNASE Inhibitor and RNA Modifying Enzymes
ScriptGuard™ RNase Inhibitor
EPICENTRE’s recombinant ScriptGuard™ RNase Inhibitor provides reliable protection for valuable
RNA samples by binding strongly to RNase A–type ribonucleases in a 1:1 ratio.
• A potent affinity for RNases (Ki >10-14 M) ensures rapid inhibition even when trace amounts of
RNase are present.
• Free of detectable RNase or DNase activity, and mammalian DNA.
• Does not interfere with enzymes commonly used to prepare or analyze RNA.
• Less sensitive to oxidation than traditional RNase inhibitors.
Cat. #
Quantity ScriptGuard™ RNase Inhibitor
SRI6325
SRI6310K
2,500 Units
10,000 Units
Thermostable RNA Ligase
Thermostable RNA Ligase catalyzes the intra- and intermolecular ligation of single-stranded RNA
(ssRNA) molecules that have a 5′-monophosphate and a 3′-hydroxyl end. The enzyme can be used for:
• Ligation-tagging of 5′-monophosphorylated RNA.
• Generating 5′-tagged RNA for RACE or other application.
• Circularization of RNA molecules.
Cat. #
Concentration
Thermostable RNA Ligase
TRL8101K
[email protected]
Quantity 50 U/µl
1,000 Units
T4 RNA Ligase is also available
LR5010
5 U/µl
LR5025
5 U/µl
1,000 Units
2,500 Units
9
RNA Modifying Enzymes
Tobacco Acid Pyrophosphatase
Tobacco Acid Pyrophosphatase (TAP) efficiently removes the cap structure present at the 5′ end of
most eukaryotic mRNAs, viral RNAs, and small capped RNAs. The end-product of a TAP reaction is a
5′-phosphorylated RNA. TAP can be used to:
• Prepare 5′-capped RNAs for 5′-ligation tagging reactions.
• Prepare template for 5′-RACE.
• Characterize the 5′-end structure of RNAs.
OH
GpppG
Capped RNA
TAP Treatment
OH
pG
Decapped RNA
Figure1. Tobacco Acid Pyrophosphatase removes the
5’-cap structure from eukaryotic mRNAs generating
RNA Ligase a
5’-phosphorylated RNA.
pG
Cat. #
Concentration
Tobacco Acid Pyrophosphatase
T81050
5 U/µl
T19050
10 U/µl
T19100
10 U/µl
T19250
10 U/µl
T19500
10 U/µl
Includes 10X Reaction Buffer.
Ligated RNA
Quantity +
pG
50 Units
50 Units
100 Units
250 Units
500 Units
pG
OH
+
Additional ligation
products
RNA 5’ Polyphosphatase
RNA 5′ Polyphosphatase,* discovered and characterized by EPICENTRE scientists, sequentially
removes the γ- and β-phosphates from 5′-triphosphorylated RNA (such as uncapped primary RNA
transcripts):
5′ pppN—OH 3′ ➞ 5′ pN—OH 3′ + 2 Pi
Unlike Tobacco Acid Pyrophosphatase, RNA 5′ Polyphosphatase has no activity on RNA with a 5′ cap
(e.g., 5′ m7GpppN—OH 3′). RNA 5′ Polyphosphatase can be used to:
• Prepare uncapped primary transcripts for 5′-ligation-tagging reactions.
• Prepare template from uncapped primary transcripts for 5′-RACE.
• Characterize the 5′ terminus of RNA molecules.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
Cat. #
Concentration
RNA 5′ Polyphosphatase
RP8092H
Includes 10X Reaction Buffer.
10
20 U/µl
Quantity 200 Units
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Ribonucleases
RiboShredder™ RNase Blend
RiboShredder™ RNase Blend is a cocktail of potent, nonmammalian RNases that completely degrades
RNA.
• Complete removal of RNA from genomic DNA preparations.
• Completely degrades RNA to mononucleotides.
RiboShredder™
RNase Blend
➞
1
2
3
4
5
6
M
Figure 1. Comparison of RNAdegrading capability of RiboShredder™
RNase Blend versus commonly-used
individual RNases or other RNase
cocktails. Nucleic acids from a standard
alkaline lysis plasmid preparation were
treated as follows under standard
reaction conditions: Lane 1, RNase A;
lane 2, RNase I; lane 3, RNase T1, lane
4, RiboShredder RNase Blend; lane
5, RNase A/RNase T1 cocktail; lane 6,
untreated alkaline lysis plasmid prep;
lane M, supercoiled DNA ladder.
Cat. #
Concentration
RiboShredder™ RNase Blend
RS12100
RS12500
1 U/µl
1 U/µl
Quantity 100 Units
500 Units
E. coli RNase H
E. coli RNase H specifically degrades the RNA in a DNA:RNA hybrid without affecting dsDNA, ssDNA,
dsRNA, or unhybridized RNA. E. coli RNase H is commonly used to synthesize ds cDNA from RNA.
Cat. #
E. coli RNase H
R52250
R0601K
Concentration
Quantity 10 U/µl
10 U/µl
250 Units
1,000 Units
Additional RNases from EPICENTRE
Hybridase™ Thermostable RNase H, RNase T1, RNase R, RNase A, RNase I (E. coli) and RNase III
(E. coli). Please visit www.EpiBio.com for details.
[email protected]
11
Ribonucleases
Terminator™ 5’-Phosphate-Dependent Exonuclease
Terminator™ Exonuclease is a 5′→3′ processive exonuclease that degrades RNAs with a 5′ monophosphate,
such as large rRNAs. RNAs with a 5′-cap structure, such as eukaryotic mRNA, or a 5′ triphosphate,
such as prokaryotic mRNAs, are resistant to Terminator Exonuclease digestion.
• Selectively digests prokaryotic 16S and 23S rRNAs, and eukaryotic 18S and 28S rRNAs, in a total
RNA preparation.
• A simple 1-hour enzymatic reaction enriches for prokaryotic or eukaryotic mRNA without the need
for columns, resins, or beads.
• Not inhibited by commonly used RNase inhibitors.
Terminator™
Exonuclease
1 Hour
Large
rRNA
mRNA
Figure 1. A 1-hour Terminator™ Exonuclease reaction digests
the large rRNAs in a eukaryotic or prokaryotic total RNA
sample, producing an enriched mRNA preparation.
B
A
Figure 2. Normal rat kidney (NRK) total RNA before (A) and after (B) Terminator™ Exonuclease
treatment. The Terminator Exonuclease-treated RNA was concentrated 10-fold.
Cat. #
Concentration
Quantity Terminator™ 5′-Phosphate-Dependent Exonuclease
TER51020
1 U/µl
20 Units
(1 Unit enriches mRNA from up to 10 µg of total RNA)
Includes Terminator™ Exonuclease 10X Reaction Buffer.
12
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cDNA Synthesis
MonsterScript™ Reverse Transcriptase
MonsterScript™ 1st-Strand cDNA Synthesis Kit
• Capable of producing full-length cDNA greater than 15 kb.
• Thermostable: retains high activity to >50°C.
• Completely lacks RNase H activity (RNase H−).
• Efficiently produces cDNA from picogram amounts of total RNA.
• The MonsterScript™ 1st-Strand cDNA Synthesis Kit includes a convenient PreMix containing
betaine* to reduce pausing and stops observed with other reverse transcriptase enzymes.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
~ 15.2 kb HGNEFp532 mRNA
5’
AAAAA . . . -3’
1.3 kb
PCR
i470407ms
A
B
kb
1.6—
1.0—
Figure 1. MonsterScript™ Reverse Transcriptase produces
full-length cDNA from mRNA greater than 15 kb. The ≈15.2-kb
HGNEFp532 mRNA was reverse-transcribed from total HeLa
RNA in a standard MonsterScript reaction. Two microliters of
the reaction was used to PCR-amplify a 1.3-kb region within 68
bases of the 5′ end of the HGNEFp532 mRNA (A). Agarose gel
electrophoresis of the 1.3-kb amplicon from the 5′ end of the
mRNA demonstrates full-length cDNA synthesis (B).
Cat. #
Quantity MonsterScript™ Reverse Transcriptase
MSTA5110
10 Reactions
MSTA5124
24 Reactions
Includes MonsterScript™ 5X Reaction Buffer.
MonsterScript™ 1st-Strand cDNA Synthesis Kit
MS040910
10 Reactions
MS041050
50 Reactions
Contents: MonsterScript™ Reverse Transcriptase (50 U/µl) with RNase
Inhibitor, MonsterScript™ 5X cDNA PreMix (contains buffer, dNTPs,
and betaine*), V3-Oligo(dT)21 Primer (10 µM), Random 9-mer Primers,
RNase-Free Water.
[email protected]
13
Related Products
CircLigase™ II ssDNA Ligase
CircLigase™ II ssDNA Ligase* is a thermostable enzyme that catalyzes circularization of singlestranded DNA (ssDNA) templates, including cDNA, having a 5′-phosphate and a 3′-hydroxyl group.
The enzyme circularizes ssDNA molecules >15 bases, including cDNAs, without the need for oligo
“bridges,” “splints” or T4 DNA Ligase. Under standard conditions, virtually no linear or circular
concatamers are produced.
CircLigase™ II ssDNA Ligase can be used for:
• Production of ssDNA templates for rolling-circle replication or rolling-circle transcription
experiments.
• Production of ssDNA templates for RNA polymerase and RNA polymerase inhibitor assays.
The enzyme offers the following benefits:
• It circularizes ssDNA molecules >15 bases, including cDNAs, without the need for oligo bridges or
splints.
• Virtually no linear or circular concatamers are produced under standard reaction conditions.
*Covered by issued and/or pending patents. See www.EpiBio.com/legal.
1
2
3
4
– circular
bp
70 –
5′ - adenylated
intermediate
50 –
linear
40 –
Exo I & III
+
+
CircLigase™ II Ligase
−
+
bp
12,216—
506—
M
Figure 1. CircLigase™ II ssDNA Ligase
converts linear ssDNA into closedcircular ssDNA. A 71-base ssDNA oligo
was converted to a circular DNA form
in a reaction containing MnCl2 and
CircLigase II ssDNA Ligase. Lane 1, DNA
markers; lane 2, 71-base linear ssDNA
oligo; lane 3, circularization proceeds
through an adenylated intermediate;
lane 4, closed-circular ssDNA reaction
product.
Figure 2. Preparation of a circular
ssDNA library. 5′-end-tagged and
fragmented T7 D111 genomic DNA was
heat-denatured and then circularized by
incubating in 33 mM Tris-acetate pH 7.6,
66 mM KOAc, 2.5 MnCl2, and 1 M betaine
for 2 hours at 60°C in the presence
or absence of 400 U of CircLigase™ II
enzyme, as indicated. Reaction products
were incubated with exonuclease I and
exonuclease III to digest linear DNA
prior to PCR amplification with primers
complementary to each tag. Reaction
products were visualized by SYBR® Gold
staining.
220—
Cat. #
Concentration
Quantity CircLigase™ II ssDNA Ligase
CL9021K
100 U/µl
CL9025K
100 U/µl
1,000 Units
5,000 Units
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Related Products
RNA Amplification Kits
TargetAmp™ RNA Amplification Kits for Expression Microarray Studies
The TargetAmp™ RNA Amplification Kits are the most robust kits for producing labeled target aRNA
(also called cRNA) from small samples for transcription profiling or expression microarray studies.
Features include:
• High yield/low input of RNA.
• Fast reactions.
• Linear RNA amplification process.
• High-quality microarray results on Affymetrix® GeneChip®, Illumina® BeadArray and other
commercial and spotted arrays.
• Evaluation kit program is available.
TargetAmp™ 1-Round RNA Amplification Kit reactions are single-tube reactions that can be
completed in about 6 hours. The TargetAmp 1-Round Kit reactions produce microgram quantities of
aRNA from 25 to 500 ng of input total RNA.
TargetAmp™ 2-Round RNA Amplification Kits are the only RNA amplification kits that enable
microarray studies from a single cell. The TargetAmp 2-Round Kit reactions produce microgram
quantities of aRNA from 10 to 500 pg of input total RNA.
MessageBOOSTER™ cDNA Synthesis Kits For Small-Sample RT-PCR Studies
The MessageBOOSTER™ cDNA Synthesis Kits amplify the poly(A) RNA contained in a total RNA
preparation and then convert the amplified RNA to cDNA that is ready for PCR or qPCR.
• Reproducibly detect even low-abundance transcripts from as little as one cell.
• Get hundreds of qRT-PCRs from as little as one cell.
• Collect samples less often and archive valuable samples.
• One-day reaction.
• Linear RNA amplification process preserves the relative abundance of transcripts present in the
sample.
Three MessageBOOSTER Kits are available that enable the researcher to work with purified total RNA,
compromised or partially degraded total RNA, or to perform a MessageBOOSTER cDNA synthesis
reaction directly from a cultured-cell lysate without having to purify RNA.
RiboMultiplier™ Sense-RNA Amplification Kit
The RiboMultiplier™ Kit utilizes a unique terminal tagging process to produce high yields of amplified
sense RNA (sRNA).
• Produce micrograms of sense RNA from as little as 10 ng of input total RNA.
• Sense RNA produced has excellent 3′/5′ ratio.
• Sense RNA produced demonstrates excellent correlation on microarrays with MAQC TaqMan® data.
Visit www.Epibio.com/RNA_Amp for additional information, and pricing for all RNA amplification
kits.
[email protected]
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