Download VeriScript™ Reverse Transcriptase

VeriScript™ Reverse Transcriptase
VeriScript Reverse Transcriptase is a mutated enzyme with
reduced RNase H activity. This enzyme is a high performing
RNase H - Reverse Transcriptase for use in a range of applications.
n Active over a wide temperature range. Recommended incubation temperature of 42°C, but incubation temperatures up to
50°C may be used for RNA templates with difficult regions
containing secondary structure or GC-rich regions.
n Robust cDNA synthesis is achieved with templates as long as
12.5 kb
n Compatible with oligo dT, random, or gene-specific priming
n Picogram level sensitivity in RT-end-point PCR
n Efficient amplification in RT-real time PCR from as few as 5
copies of synthetic RNA
Source:
Recombinant
Purity:
Greater than 95% pure as determined by SDS-PAGE.
Tested for contaminating endonucleases, exonucleases, and
ribonucleases.
Unit definition:
One unit is the amount of enzyme activity that incorporates 1
nmole of dTTP into acid insoluble material in 10 minutes at 37°C
using poly(rA)-oligo(dT)18 as template-primer.
Functional test:
Functionally tested to synthesize cDNA from total RNA to generate a 10.9 kb product by RT-PCR.
Properties:
VeriScript Reverse Transcriptase is an engineered version of
M-MLV Reverse Transcriptase with reduced RNase H activity and
increased thermostability, allowing for first-strand cDNA synthesis with more full length cDNAs up to 12.5 kb. This DNA polymerase can synthesize complementary DNA from single-stranded
DNA, RNA, or a RNA:DNA hybrid. VeriScript is active up to 50°C
which offers greater sensitivity and consistent performance for a
wide range of RT applications.
Concentration:
200 units/µl
Buffer:
Functionally tested 5X VeriScript RT Reaction Buffer
(included with enzyme):
250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2.
Fig. 1. Increased thermostability
0.5 kb
M
42
45
48
2.8 kb
50
55 42
45
48
6.6 kb
50
55
42
45
48
amplicon length
50
55
M
T (°C)
12.0 kb 4.0 kb 2.0 kb -
1.0 kb 500 bp -
100 bp -
HeLa total RNA (1 µg) was used in a 20 µl first-strand cDNA synthesis with 200 units of VeriScript Reverse Transcriptase. Reactions were incubated at the temperatures indicated on the figure for 30 minutes, followed by heat inactivation for 15 minutes at 70°C. RNA was removed by adding 1 µl (5 units) RNase H
(PN 70054) and incubation at 37°C for 10 minutes, followed by heat inactivation for 10 minutes at 65°C. 10 ng cDNA was used in a 50 µl PCR reaction using
FideliTaq™ DNA Polymerase (PN 71180) for 35 cycles. VeriScript Reverse Transcriptase is active over a wide temperature range. Incubation temperatures up to
50°C may be used for RNA templates with difficult regions containing secondary structures or GC-rich regions.
Components:
VeriScript Reverse Transcriptase 4,000 units
1 ml 5X VeriScript RT Reaction Buffer
1 ml DTT
Storage:
Shipped on dry ice. Store at -20°C.
VeriScript Reverse Transcriptase
VeriScript Reverse Transcriptase 10,000 units
1 ml 5X VeriScript RT Reaction Buffer
1 ml DTT
Product code
Pack size
78070
4,000 units
10,000 units
VeriScript Reverse Transcriptase, 4 x 10,000 units
4 x 1 ml 5X VeriScript RT Reaction Buffer
2 x 1 ml DTT
4 x 10,000 units
Fig. 2. Sensitivity in RT-real time PCR
40
PCR efficiency = 96.7%
R2 = 1.00
35
Ct
30
25
20
15
10
1
2
3
4
5
6
7
8
9
Log starting quantity
Decreasing amounts of B. subtilis DAP RNA (10 8 to 102) molecules were converted into cDNA in the presence of 1 ng HeLa total RNA using 50 units of VeriScript
Reverse Transcriptase in a total reaction volume of 20 µl. 1/20 th of the cDNA was used in USB VeriQuest® SYBR® Green Real-Time PCR (PN 75600). Efficient
amplification was shown from as few as 5 copies of DAP mRNA.
Fig. 3. Picogram level sensitivity in end-point RT-PCR
1 pg
10 pg
100 pg
1 ng
10 ng
M
1 µg
1 pg
10 pg
100 pg
1 ng
10 ng
100 ng
1 µg
M
100 ng
SuperScript II
VeriScript RT
Total RNA
2.0 kb -
500 bp -
50 bp -
Decreasing amounts of HeLa total RNA (1 µg to 1 pg) were used in a 20 µl first-strand cDNA synthesis reaction with 200 units of VeriScript Reverse Transcriptase
or SuperScript® II. Reaction was incubated at 42°C for 30 minutes, followed by heat inactivation for 15 minutes at 70°C. 1/10 th of the cDNA was used to amplify
a 0.5 kb target (GAPDH) by VeriQuest Taq DNA Polymerase (PN 71170) in a 50 µl PCR reaction for 40 cycles. Sensitivity of VeriScript Reverse Transcriptase is
comparable to that of SuperScript II.
For Research Use Only. Not for use in diagnostic procedures.
Affymetrix, USB, and VeriQuest are registered trademarks of Affymetrix, Inc. VeriScript and FideliTaq are trademarks of Affymetrix, Inc. Taq DNA Polymerase—sold under licensing arrangements
with Applied Biosystems. Purchase is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated
performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. SYBR is a registered trademark of
Molecular Probes, Inc. and is provided under an agreement with Molecular Probes, Inc. All other trademarks are the property of their respective owners.
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Rev 08/03/15