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Protocol for First-strand cDNA Synthesis

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Protocol for First-strand cDNA Synthesis
PCR template generation (with M-MuLV Reverse Transcriptase)

Prepare in a sterile tube:
- template RNA:
  total RNA 0.1-5µg
  or poly(A)⁺mRNA 10ng-0.5µg,
  or specific RNA 0.01pg-0.5µg
- primer:
  oligo(dT)₁₈ 0.5µg
  or random hexamer 0.2µg,
  or sequence-specific 15-20pmol,
- deionized water (nuclease free) up to 11µl.
Incubate the mix at 70°C for 5 minutes and chill on ice.
Add the following in the order indicated:
- 5X reaction buffer 4µl,
- 10mM 4 dNTP mix 2µl (1.0mM - final concentration),
- ribonuclease inhibitor 20u,
- deionized water (nuclease free) to 19µl.
Incubate at 37°C for 5 minutes. If random primer is used, incubate at 25°C for 5 minutes.
Add 40 units of M-MuLV Reverse Transcriptase. Incubate the reaction mixture, containing oligo(dT)₁₈ or sequence-specific primer at 37°C for 60 minutes. If using random hexamer primer, incubate at 25°C for 10 minutes and then at 37°C for 60 minutes.
Stop the reaction by heating at 70°C for 10 minutes. Chill on ice.

Note

The synthesized cDNA can be amplified by the PCR (see Protocols for PCR using Taq and Pfu DNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.

Reference

Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.

Ordering Information

M-MuLV Reverse Transcriptase #EP0351
#EP0352 1000u (20-40u/µl)
5000u (20-40u/µl)

10mM dNTP Mix #R0191
#R0192 0.2ml
1.0ml

Ribonuclease Inhibitor #EO0311
#EO0312 1000u (20-50u/µl)
5x1000u (20-50u/µl)

First Strand cDNA Synthesis Kit #K1611
#K1612 for 10 reactions
for 100 reactions

Taq DNA Polymerase (recombinant) #EP0401
#EP0402
#EP0403
#EP0404
#EP0405
#EP0406 100u (5u/µl)
500u (5u/µl)
LC, 100u (1u/µl)
LC, 500u (1u/µl)
5x500 (5u/µl)
10x500u (5u/µl)

Taq DNA Polymerase (native, without BSA) #EP0281
#EP0282
#EP0283
#EP0284 100u (5u/µl)
500u (5u/µl)
LC, 100u (1u/µl)
LC, 500u (1u/µl)

Taq DNA Polymerase (native, with BSA) #EP0071
#EP0072 100u (5u/µl)
500u (5u/µl)

Pfu DNA Polymerase (native) #EP0571
#EP0572 100u (2.5u/µl)
500u (2.5u/µl)

Pfu DNA Polymerase (recombinant) #EP0501
#EP0502 100u (2.5u/µl)
500u (2.5u/µl)

2X PCR Master Mix #K0171 for 100 reactions

Deoxyribonuclease I (RNase free) #EN0521 1000u (1u/µl)

DEPC-treated Water, molecular biology grade #R0601 30ml

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Updated August 23, 2002 13:46