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(MuLV Reverse transcriptase, RNase H minus)

Catalog #Pack sizePrice, EuroSHOP
10510010000 u85,00Add to cart
10525050000 u295,00Add to cart

 

Datasheet: Reverase (M-MuL V)

Comparison/Test of Reverase (M-MuL V)

Description:
<em>Reverase</em>TM is M-MuLV Reverse transcriptase purified from <em>E.coli</em>  strain harbouring a plasmid that directs the synthesis of modified form of Moloney Murine Leukemia virus (M-MuLV) reverse transcriptase. M-MuLV reverse transcriptase is an RNA or DNA directed DNA polymerase. The enzyme can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single stranded DNA as a template. This enzyme had been genetically altered to remove associated RNase H activity. Removal of the RNase H activity resulted in an increase of full-length cDNA products. MW of Reverase is 69 KDa.

Concentration:
200000 - 400 000 units/ml

Storage buffer:
50 mM TrisHCl, pH8.3, 100 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 0,1%Triton X-100, 50% glycerol.

Recommended reaction buffer for RT-PCR (1x) :
50 mM TrisHCl (pH 8.3 at 25oC), 2-8 mM MgCl2, 10 mM DTT, 100 mM KCl (Optional: 2-4 mM MnCl2)

Supplied 5xRT buffer "complete": 250 mM TrisHCl, pH 8.3; 500 mM KCl, 15 mM MgCl2, 50 mM DTT  

Supplied 5xRT buffer "incomplete": 250 mM TrisHCl, pH 8.3; 500 mM KCl and with  extra tubes MgCl2 (100 mM) and DTT (100 mM)

Unit definition:
One unit of activity is the amount of enzyme required to incorporate 1 nmole of dTTP into an acid-insoluble form in 10 minutes at 37oC using polyA-oligo(dT) as template and primer.

Storage conditions: -20oC  

cDNA synthesis with Reverase

Protocol

1. Mix in the tube: 1-5 µg of the total RNA (or 50-500 ng of polyA RNA) 10 pmole of strand-specific primer (or 250-500 ng of oligo-dT for each µg of RNA) add water up to 8 µl

2. Incubate the mixture 10 min at 70°C, then 10-15 min at room temperature (for the specific primer) or place in ice in the case of oligo dT or random primer

3. Add into the mixture:

-  4 µl of 5xRT buffer complete (250 mM TrisHCl, pH 8.3; 500  mM KCl, 15  mM MgCl2, 50
    mM DTT)
- 1 µl of dNTP mix (10 mM of each dNTP; Cat.-No: 110001 and 110002)
- RNAsin - 20-40 units (optional)
- Reverase - 200 units
- H2O - up to 20 µl

4. Incubate the mixture at 37-55°C during 30-120 min. The time of reaction depends on the length of cDNA, 30 min if enough for cDNA in range of 500 bp in length, 120 min is for cDNA more then 1.5 kb. The temperature of the reaction depends on the structural features of RNA. Use increased temperature (up to 55°C) for the highly structured RNA. The optimal temperature and reaction time should be adjusted for each particular RNA. We recommend to use buffer with pH 8.8 if the reaction is performed at elevated temperature.

5. Heat the mixture 10 min at 65-70°C to inactivate the Reverase.

6. Use the mixture for PCR or for other application. Reverase and RTx5 buffer are free from RNase activities, meanwhile we recommend to add RNasin into the mixture to inhibit possible RNase contaminations of the sample.                      

For your PCR-Reaction you need  5-10 µl of your RT-PCR product. Find the standard protocol for PCR in our Web-Page: PCR Theory Section.

<em>Please add the appropriate amounts of MgCl2, MnCl2 (optional), DTT, dNTPs for the reaction.</em>

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