Reverse Transcription PCR
RNA -> LOTS OF DNA
(modified from PCR Protocols January 1991 )
Ed Rybicki
copyright 1992, 1996
Contents
Reverse Transcription Reaction
Reverse Transcription Reaction:
This provides the cDNA - by extension from a primer complementary to the RNA sequence - for the amplification by PCR using 2 primers.
REMEMBER TO USE A PRIMER WHOSE SEQUENCE IS COMPLEMENTARY TO THE RNA!!
Reverse Transcription (RTase) Reaction Mix:
It is convenient to make up a single master mix to be aliquotted out for a number of RNAs to be reverse transcribed using the same primer.
sample mix: per 10ul
- 2ul RNA preparation
- 1 ul DMSO
- 1 ul 40 U/ul RNAsin
- 6ul DEPC-H20 per reaction:
NB: master rxn mix made separately!!
Total volume master rxn mix:
- 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!!
Mix everything together, leave on ice
HEAT SAMPLE MIX AT 65 C FOR 3 MIN, --> WET ICE
ADD 10ul RTase MIX / SAMPLE
Polymerase Chain Reaction (modified from PCR Protocols)
Reaction Mix:
It is again convenient to make up a master mix to be aliquotted out to amplify all samples, if they are to use the same primers. If not, modify master mix by simply leaving out primers.
Total volume: 50ul / rxn x N + 5-10% for wastage
Reaction master mix:
- 1/10 vol Cetus/ Promega /other 1 0x buffer
- 1/50 - 1/25 vol 2.5mM stock dNTPs [--> 50-100uM]
- 1/20 vol 10uM forward (=RNA sense) primer [--> 0.5uM]
[NB: no reverse primer needed IF THIS IS THE.SAME AS cDNA PRIMER as
residual cDNA synthesis primer concn. is +/- 5uM]
- OPTIONAL: 1/20 - 1/10 vol DMSO
- 0.5ul of 5U/ul Taq polymerase / 100ul rxn mix
REMEMBER TO ALLOW FOR 5ul/rxn OF SAMPLE!!
- MIX MASTER RXN MIX, LEAVE ON ICE.
- Aliquot out 45ul / PCR rxn vial
- Add 5ul sample / vial from reverse transcription reaction mix
- Add 50ul / vial mineral oil (NB: new upipette tips each time!!)
- Vortex lightly, spin down
PCR Conditions: recommended:
- 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