Method adapted from RRG method by Janelle Hoskins,
Department of Pharmacology, University of Sydney
2nd February, 2004
PCR for semi-quantifying GAPDH cDNA (housekeeping gene)
Application:
Semi-quantifying GAPDH mRNA from cell line studies.
Manufacturer:
Qiagen for Taq polymerase and buffer, MBI (Progen) for dNTPs.
Samples:
cDNA prepared using CCIA method (random hexamers).
Thermocycler:
Eppendorf (Nongrad TC) in RRG
Primers:
Primer GAPDH Fwd (from RRG, stock: 100 µM)
Primer GAPDH Rev
Master Mix can be prepared and mixed at room temperature.
In a 0.5 mL thin-walled PCR tube, prepare a Master Mix of 1.1 to 1.3X adding the following
reagents (without the template):
1X
Qiagen PCR Buffer
1.0
dNTPs (2 mM)
1.0
Primer F (10 µM)
0.125
Primer R (10 µM)
0.125
Autoclaved Milli-Q water
6.55
Hotstar Taq
0.2
Template (cDNA)
1.0
10 µL
Pipette 9 µL Master Mix into a 0.5 mL thin-walled PCR tube. Add 1 µL template (~ 25 ng total
RNA) to tube. Mix by flicking tube and briefly centrifuge to bring down droplets from the side
of the tube.
Thermocycler program:
1. 95oC 15 min
2. 94 oC 45 sec
3. 65 oC 15 sec
4. 72 oC 30 sec
5. go to #2, repeat 21
6. 72 oC 5 min
7. hold 4 oC
Method adapted from RRG method by Janelle Hoskins,
Department of Pharmacology, University of Sydney
2nd February, 2004
Visualising results
Weigh 2.0 g agarose into a 500 mL Schott bottle. Add 50 mL 1X TAE buffer. Microwave bottle
for 1 min on high (without lid). Swirl bottle to mix agarose. Microwave bottle for a further 10
sec on high. Swirl bottle to mix. Rotate the bottle under running cold water until bottle is cool.
Add 2 µL 5 mg/mL ethidium bromide to agarose and swirl the bottle to mix. With masking tape
and comb, prepare the gel case for casting. Pour the agarose into the gel case. When the gel has
set, remove the comb and masking tape. Place the gel into the electrophoresis tank and submerge
it in 1X TAE.
In lane #1, pipette 4 µL 50 bp ladder (MBI Fermentas, Progen). Load 6 µL of digested samples
(10 µL digested sample + 1 µL 6X loading buffer) into the other lanes. Apply 70 V and run the
gel for 60 min.
Example gel (02/02/2004):
Expected size of amplicons: ~500 bp
Lane 1: 50 bp marker