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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)

  1. Prepare in a sterile tube:
  2. Incubate the mix at 70°C for 5 minutes and chill on ice.
  3. Add the following in the order indicated:
  4. Incubate at 37°C for 5 minutes. If random primer is used, incubate at 25°C for 5 minutes.
  5. 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.
  6. Stop the reaction by heating at 70°C for 10 minutes. Chill on ice.

 Note 

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