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!!
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:
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:
Mix everything together, leave on ice
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.
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
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
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