PT-PCR

REVERSE TRANSCRIPTION PCR:

RNA -> LOTS OF DNA

Contents

Reverse Transcription (RTase) Reaction Mix:

• 2ul RNA preparation
• 1 ul DMSO
• 1 ul 40 U/ul RNAsin
• 6ul DEPC-H20 per reaction:

NB: master rxn mix made separately!!

• 22ul X N (no. samples) + 5% 1/10 total vol
• 1 OmM dNTPs
• 1/5 vol BRL 5x RTase buffer [MuLV enzyme]
• 1/20 vol 20 U/ul MMuLV RTase [NB: dilution of 200U/ul stock ! !]
• 0.5 ug specific primer [eg. 0.3ul of 288uM=1.9ug/ul 20-mer]
• 1/20 vol DMSO [dimethyl sulphoxide]
• DEPC-H20 to 22ul/rxn:

REMEMBER TO ALLOW FOR 10ul/rxn FOR SAMPLE!!

HEAT SAMPLE MIX AT 65 C FOR 3 MIN, --> WET ICE

ADD 10ul RTase MIX / SAMPLE

MIX BY VORTEXING, HEAT SAMPLES AT 42 C 60 min / 52 C 30 min

:

• 94o C 3 min; 45-50oC 3 min; 72oC 3 min;
• (93oC 1 min; 45-50oC 1 min; 72oC 1-3 min) x 30-34 cycles
• 72oC 5-10 min