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商品編號


RG90925



品牌


Lucigen



公司


Lucigen



公司分類(lèi)


Reverse Transcriptase Enzymes



Size

2,500 Units





商品信息

An optimized reverse transcriptase system for the production of full-length
CDN
A.

Produce full-length
CDN
A with RNAse H- mutant

Achieve higher specificity at elevated temperatures, up to 55°C




EpiScript? Rnase H
-
Reverse Transcriptase (EpiScript RT), an alternative to SuperScript? II Reverse Transcriptase, is a recombinant MMLV reverse transcriptase with greatly reduced RNase H activity. It is highly efficient at producing full-length
CDN
A from long RNA templates. EpiScript RT is capable of producing
CDN
A from as little as 50 pg of total RNA for real-time RT-PCR (qRT-PCR) analysis and other applications.

Applications

First-strand
CDN
A synthesis for subsequent PCR or real-time PCR.

Benefits

Recombinant MMLV reverse transcriptase with greatly reduced RNase H activity

Active at temperatures up to 55°C

Highly efficient at producing full-length
CDN
A from as little as 50 pg of total RNA

Best value in an RNase H- Reverse Transcriptase

Storage:
Store only at -20°C in a freezer without a defrost cycle.

Storage Buffer:
EpiScript RT is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton? X-100.

Unit Definition:
One unit of EpiScript RT catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using saturating amounts of oligo(dT)-primed poly(rA) as template.

Contaminating Activity Assays:
EpiScript RT is free of detectable exonuclease, endonuclease, and RNase activities.

Figure 1. EpiScript? Reverse Transcriptase performed equally or better than comparable reverse transcriptases from other vendors.
First- strand synthesis reactions were assembled according to manufacturer?s specifications. Input RNA was 1 ?g of Jurkat total RNA (
Ambion
?). Reactions were primed using 50 ng of poly-T
(16-18)
DNA. 2nd strand qPCR was performed using
Bio-Rad
iQ SYBR mastermix and gene-specific primers that yielded 250-350 bp amplicons. Reactions were repeated 4-fold. PGDF-R (Platelet Derived Growth Factor Receptor), TNF (Tumor Necrosis Factor), IL-1b (Interleukin-1 beta), IL-2 (Interleukin 2). Image courtesy of Matthew Kellinger,
Illumina
? Inc.




Figure 2 (click to enlarge). EpiScript? Reverse Transcriptase produces similiar transcript coverage independent of transcript length.
EpiScript was used to prime first strand
CDN
A either from total RNA using oligo-dT (left panel) or polyA+ selected RNA with random hexamers (right panel). The
CDN
A was converted into
Illumina
?-compat
IBL
e libraries and sequenced. The reads were aligned to transcripts based upon various length classes and read density plotted relative to the percent distance from the 5? end of the transcripts; 0% refers to the 5? end and 100 % is the 3? end. As expected, oligo-dT priming results in a more pronounced 3? bias than random priming.


Figure 3. Use of EpiScript? Reverse Transcriptase for first strand
CDN
A results in detection of similar transcript categories independent of input amount.
EpiScript RT was used to random prime 5 ng or 50 pg of human total RNA and the
CDN
A was converted into libraries for
Illumina
? sequencing. Reads were aligned using Tophat and annotated with Cufflinks and the percentage of each major category is visualized as a pie chart. Equivalent results are observed for both 5 ng and 50 pg of input RNA.


ORDER INFORMATION
Contents: EpiScript RNase H
-
Reverse Transcriptase, 10X Reaction Buffer, 100 mM DTT.