PCR Protocol
Jenny, January 29th, 2004.
Test that your primers do not amplify DNA.
Mix 1μg RNA with
Add primer 0.5μl forward
Add primer 0.5μl reverse
Add dNTP 1μl
Add dH2O Water to total 19 μl
Add 1μl Taq polymerase
PCR
2 min 94ºC to denature
Then 30-35 cycles
Denature 45 sec 94ºC
Anneal 45 sec 55ºC
Polymerize 75sec 72ºC
Run resulting reaction mix on TAE gel – should see nothing (or some primer dimer given
the high concentrations).
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PCR Protocol
Jenny, January 29th, 2004.
2-step RT-PCR with SybrGreen
cDNA synthesis
Use BD biosciences advantage RT-PCR kit (Cat K1402-1 for 25 reactions, K1402-2 for
100 reactions).
Book PCR machine.
Clean the bench and pipettes with ethanol.
Use only RNase free tips and tubes.
Make master mix for samples, in individual PCR tubes.
If only have one or two, add components individually to RNA, adding MMLV last.
 4μl 5 times reaction buffer
 1μl dNTP mix (10mM each)
 0.5μl RNase inhibitor
 1.0μl MMLV reverse transcriptase
Vortex a lot, and spin down.
Total volume is 6.5μl per sample.
In clean PCR tubes, add
 0.2-1.0 μg of RNA made up to 12.5μl with ultraspec sterile H2O (or DEPC H20)
 1μl of random hexamers OR oligo dT primers
 6.5 μl of the above master mix.
 Mix by pipetting up and down.
TOTAL VOLUME should be 20μl.
PCR machine
 42ºC for 1hr.
 94ºC for 5 min (stop reaction and destroy DNase activity).
 4ºC to cool.
Spin down.
Dilute to desired final volume (I make with 1μg RNA and dilute final to 250μL and use
5mcl per PCR well).
Transfer to sterile eppendorf and store at –80ºC until use. If planning to use same sample
on a lot of plates aliquot before freezing to avoid repeated freeze thaws.
PCR Protocol
Jenny, January 29th, 2004.
RT-PCR for SybrGreen
Book Light cycler!!!!!!
Clean bench and pipettes with EtOH.
Sterile tips.
Get optical plate and caps.
Keep plate on ice.
Try to plan experiments to allow leaving one no-cDNA well for each pair of primers
(shows up contamination of your primers).
Make a primer mini-master mix (this one is for 24 samples using same primer (8 in trip
or 12 in duplicate, but adjust as needed).
 0.5μl forward primer
 0.5μl reverse primer
 140μl ultraspec H20.
Vortex ++.
To plate, add experimental samples.
 Add 5μl cDNA mix to each well, if you make similar cDNA to mine.
 Add 5μl of appropriate primer mix to each well.
 Add 10μl of SybrGreen master mix per well.
Total reaction volume is 20μl.
Put on optical caps – make VERY sure that the lids are all down flat – the laser shines up
through the lid, so it must be flat.
Spin down in plate-spinner in Shoelson lab if necessary.
Put in PCR machine!
After the first run for EVERY set of primers, run the contents of the well on a gel to
make sure that you aren’t getting just primer-dimer, to make sure that you DO have a
product of the right size, and hopefully to make sure that you don’t have another product
of a different size (although this can sometimes be isoforms, rather than non-specific
products).