10 x Reaction Buffer

25 mM MgCl₂

dNTP Mix,10mM each

0.05% [w/v] Gelatin

Oligo-p(dT)15 Primer, 0.8µg/µl

Random Primer p(dN)6, 2µg/µl

RNase Inhibitor, 50 U/µl

MMLV Reverse Transcriptase

RNase-free ddH₂O

Protocol

• It is recommended that the first strand cDNA synthesis is carried out under conditions where RNase contamination has been eliminated. Pipette tips and tubes should be treated with 0.1% diethylpyrocarbonate (DEPC) (soak overnight in 0.1% aqueous solution of DEPC at 37°C, then heat at 10°C for 30 min and autoclave).
• Wearing gloves is highly recommended.
• One unit of M-MuLV RT incorporates 1nmole of deoxyribonucleotide into DE81 absorbable form in 10min at 37°C.
• One unit of ribonuclease inhibitor is defined as the amount of protein required to inhibit 50% of the activity of 5ng of RNase A.

Description:
This kit is designed for preparation of full-length first strand cDNA from RNA templates. The first strand cDNA synthesis kit relies on a cloned enzyme, Moloney murine leukemia virus reverse transcriptase (M-MuLV RT). M-MuLV RT synthesizes first strand cDNA at sites determined by the type of primer used:

1. random hexamer primer: at non-specific points along an RNA template. In this case, all RNA in a population are templates for cDNA synthesis.
2. oligo(dT)18: at the 3´-end of poly(A)+ mRNA. In this case, only mRNA with 3´-poly(A) tails are templates for cDNA synthesis.

First strand cDNA synthesized with this system can be used as a template in the polymerase chain reaction (PCR*). As the reaction conditions of cDNA synthesis and PCR* are compatible, cDNA reaction mixture can be added directly to a PCR* mixture. The kit is optimized for maximum yield of full length cDNA. The addition of ribonuclease inhibitor lowers the risk of mRNA degradation during the reaction.
The first strand cDNA synthesized may also be used as a template for second strand synthesis.

*The Polynucleotide Chain reaction (PCR) is covered by the patents owned by Hoffman-La Roche Inc.

References:

1. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual (2nd Ed.) Cold Spring Harbor University Press, Cold spring Harbor, NY, 1989.
2. Ausubel, F.M. et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, 1997.
3. Gerard, G.F., Dlessio, J.M., Meth. in Mol. Biol., V. 16, 73-93, 1993.