Description

Reverase ^TM is M-MuLV reverse transcriptase purified from E.coli 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

200 – 400 u/μl

Storage buffer

• 50 mM Tris-HCl, pH 8.3
• 100 mM NaCl
• 1 mM EDTA
• 0.1 mM DTT
• 0,1 % Triton X-100
• 50 % glycerol

Storage conditions

-20 °C

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 37 °C using polyA-oligo(dT) as template and primer.

Recommended reaction buffer for RT-PCR (1x)

• 50 mM Tris-HCl (pH 8.3 at 25 °C)
• 2 – 8 mM MgCl ₂
• 10 mM DTT
• 100 mM KCl (Optional: 2 – 4 mM MnCl ₂ )

Supplied 5x RT buffer “complete”

• 250 mM TrisHCl, pH 8.3
• 500 mM KCl
• 15 mM MgCl ₂
• 50 mM DTT

Supplied 5x RT buffer

• 250 mM Tris-HCl, pH 8.3
• 500 mM KCl
• with extra tubes: MgCl ₂ (100 mM) and DTT (100 mM)

Protocol

1. Mix in the tube: 1 – 5 µg of the total RNA (or 50 – 500 ng of polyA RNA) and 10 pmol 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 5x RT buffer complete (250 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl ₂ ; 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
• H ₂ O – 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 is for cDNA in range of 500 bp, 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 5x RT 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.

Please add the appropriate amounts of MgCl ₂ , MnCl ₂ (optional), DTT, dNTPs for the reaction.