Real Time PCR Protocol
1. Isolate your RNA using the Lithium Chloride protocol.
2. Check your RNA quantity and quality using the Nanodrop and check gel.
3. Treat the RNA with DNase 1 Amp Grade (Invitrogen):
2ug RNA sample
1uL 10X DNase 1 Reaction Buffer
1uL DNase 1 Amp Grade1U/uL)
DEPC treated water to 10uL
4. Incubate at room temperature for 15 minutes.
5. Inactivate the DNase 1 by adding 1uL of 25mM Edta to the sample.
6. Heat at 65oC for 10 minutes.
You can store your samples at -80oC at this point.
QPCR cDNA Synthesis
Using the AffinityScript QPCR cDNA Synthesis Kit (Stratagene) prepare the first-strand
cDNA synthesis by adding the following in order:
RNase-free water to a total volume of 20uL
10.0uL of first strand master mix (2X)
3.0uL of oligo(dT) primer or random primers (o.1ug/uL)
1.0uL of AffinityScript RT/RNase Block enzyme mixture
5.0uL of DNase treated RNA sample (500ng)
1. Incubate the reaction at 25oC for 5 minutes.
2. Incubate the reaction at 42oC for 15 minutes.
3. Incubate the reaction at 95oC for 5 minutes.
Place reactions on ice and add 80uL DNase free water.
The samples can be used immediately in the QPCR,or stored long-term at -20oC
Brilliant SYBR Green QPCR Master Mix
1. Switch on computer.
2. Switch on Stratagene Mx3005P (switch at rear of machine)
3. Prepare the experimental reactions by adding the following components in order:
Experimental reaction: (ON ICE)
8.0uL Nuclease-free water to adjust the final volume to 25uL (including 2.5uL of
experimental cDNA from QPCR Master Mix reaction)
12.5uL of 2x Master mix
1uL of upstream primer (50-150nM final concentration)
1uL of downstream primer (50-150nM concentration)
4. Gently mix the reactions without creating bubbles.
5. Add 2.5uL of experimental DNA to each reaction.
6. Gently spin the strip tube reactions in the small centrifuge, or if using plates use
Murray's centrifuge.
7. Give the tubes/plates a gentle tap to ensure complete mix of reagents.
8. Place the reactions in the instrument.
9. Select the MxPro icon.
10. Select quick set up.
11. Select turn lamp off at end.
12. Press Run.
N.B. Until we have been properly trained to use the software, please see Rich who will
show you how to analyze your results.