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
