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Reverse Transcriptase

by Gene And Cell Technologies
Price $80.00

Reverse Transcriptase

Reverse Transcriptase is a DNA-dependent RNA polymerase.  It produces complementary DNA (cDNA) from RNA templates.  The primary use of this enzyme is in the quantitation of gene expression using quantitative polymerase chain reaction (qPCR) and a variety of molecular biology techniques. 

Our Reverse Transcriptase is an engineered variant based on Moloney Murine Leukemia Virus (MMLV).  It bears mutations that improve its solubility, enhance its heat stability, and silence its RNAse H activity.  With that set of mutations, its activity is comparable to many commercial brands of Reverse Transcriptase. 

 

Specifications

- 1 unit polymerizes 1 nmol of dTTP under standard reaction conditions. 

- Activity >1,000 U / mg of lyophilizate

- Reaction Temperature:  42C

- RNAse < 1 milli-KU per U of RT

- DNAse not detected < 10 milli-KU per U of RT

- Protease not detected at < 1 ng Proteinase K equivalents per U of RT

- Phosphatase not detected at < 30 micro-U per U of RT

 

Storage Conditions

The lyophilized enzyme is stable at ambient temperature during shipping and short term storage.  For the longer term it should be stored in the fridge (+4C).  Once reconstituted in storage buffer (not provided, recipe below), it should be stored at -20C in liquid form.

 

Recommended Buffers

This product is designed to give you the most enzyme for the best price.  Buffers and other components are not included.  We suggest the following recipes to prepare reactions on your own:

 

Storage Buffer (1x)

50 mM Tris pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% IGEPAL-CA-630, 50% Glycerol.

 

Reaction Buffer (1x)

50 mM Tris pH 8.2, 3 mM MgCl2, 10 mM DTT.

 

Pipetting Plan

Reaction buffer (10x)...........................2 ul

dNTPs, 10 mM each............................1 ul

Random Primer d(N9), 1.5 ug/ul..........2 ul

Template RNA......................................0.02-2 ug

Water....................................................to 20 ul

RT enzyme (200U/ul).............................1ul

Many varieties of these recipes will work. If you have worked with a different MMLV-based reverse transcriptase, your existing reaction and storage conditions will likely work just fine. 

 

Typical Results

To illustrate the activity of our Reverse Transcriptase, we prepared dilutions of the enzyme:  First we weighed in 262 mg of the lyophilizate.  Then, dissolved it in 970 ul of the recommended storage buffer as above.  This would result in concentration of 0.270 mg/ul or 270 U/ul.  Then we diluted the enzyme in additional storage buffer as shown in the legend of the chart below. 

We purified total mRNA from 3 million cultured HEK293T cells using an oligo-dT bead based kit from a major US vendor.  We used a total of 80 ng of this poly-A mRNA in a reverse transcription reaction, following the pipetting plan above.  Finally, we transferred 2 ul of the resulting cDNA into a 20 ul qPCR reaction using Taq Polymerase from Gene And Cell Technologies, with EvaGreen detection dye.  PCR primers were targeting the ACTB housekeeping gene. 

=> It can be seen that the enzyme is able to transcribe the 80 ng to near completion, even at extreme dilution factors. 

While your mileage may vary to some degree, depending on your individual sequences and reaction conditions, this experiment suggests that thousands of qPCR reactions can come out of even the smallest of our vials. 

 

 

 

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