Reverse Transcription - Polymerase Chain Reaction (RT-PCR)
(Standard recipe)
I dilute all the RNA to 300ng/µl, and then RT & amplify 1µl of this per set of primers.
The primers are combined in a single solution containing 20µM of each, and 1µl of this is used in the
PCR step.
The following recipe uses only 1/4th of the volumes recommended, so that 400 reactions can be made
from each kit. It is designed for amplifying a number of RNA samples, each with a number of different
primer pairs. Several volumes of each RNA are reverse transcribed to provide one volume per primer
pair. After RT a PCR "master mix" is added, and one volume of this taken and mixed in a fresh PCR
tube with 1µl primer pair.
(i) Make an RT "master mix". This recipe is for each reaction, so if making enough for
10 reactions, multiply all the volumes by 10.
Volume of RT mix
(number of RNA samples x (number of primers + 2)) +3 = X
(the "+1" volume is to allow all of the RT mix volume to be taken!)
RT mix
MgCl2
10xPCR buffer
dNTP's
RNase inhibitor
MuLV RT
random hexamers
(per reaction)
µl *X total vol
1.0 if 25 mM stock
0.5 if 50 mM stock
0.5 or 1.0 5 X RT buffer
2.0
0.25
0.25 (perkin RT 50U/ul) 0.06 (GIBCO RT 200 U/ul)
0.25 4.25
Vol of RT mix per tube for RT step
= 4.25 x (number of primers + 1) = Y
In each PCR tube add Y µl RT mix and (number of primers + 1) µl diluted RNA
Incubation Conditions:
/42°C/95°C/20°C
for
35/ 5/ 1 min
in PCR machine for ease.
(ii) Prepare the same number of volumes of PCR "master mix"
PCR mix
µl
total
*X
MgCl2
0.5 or 0.25 if 50 mM stock
10xPCR buffer
2.12
water
16.23
Taq polymerase
0.125
Ab to Taq pol 0.05 19.0
-to each RT tube add (number of primers + 1) * 19.0µl PCR mix
-mix by pipette
-enlever 24.25µl to a fresh PCR tube containing 1µl primer pair
Incubation Conditions:
95°C/60°C/72°C
for
45s/ 1/ 90s
for 36 cycles; then 72°C 8 min (no first long 95°C step other than 30s)
I usually run for a large numlber of cycles until I see a product then cut down on the
cycles.
I usually load 10µl PCR reaction product (so I can run it again) with 4µl DNA loading
dye and run on a 2% gel.