Guys, I REALLY NEED HELP
At the end of the last year I optimized every parameter of my RAPD-PCR, and as a result I did obtain great products multiple times. Then, I went home for my vacation (I wish I didn't) and now I'm back at the lab, and guess what? I CAN'T MAKE IT WORK AGAIN.
I already: changed PCR Buffer, dNTP, water, magnesium solution, changed the primer, re-patterned my DNA samples, autoclaved every tip and tube again, tried a different thermocycler...
DOES ANYONE HAVE ANY IDEA OF WHAT'S GOING ON?
I'll let all my conditions and a picture of my previus PCR results below:
Final concentrations in the mixer (13 uL) = 2,5 mM MgCl2, 1x PCR Buffer, 800 mM primer, 30ng DNA (total), 0,2 mM dNTP, 1U Taq.
PCR run:
Hot Start at 96°C for 3'
45 cycles of 92 °C for 1', 38 °C for 1', 72 °C for 2'. (already tried with 40 cycles)
Final extension at 72 °C for 5'.
Primers I'm currently using: OPC-05 (GATGACCGCC)
What do you mean by not working?
No amplification at all or a smear on the gel?
Can we see a picture of the not working pcr please?
Is any other pcr by any other worker working on this pcr machine?
It's not amplifying. I can show you a picture latter, but it will be just a blank gel with ladder.
I'm the only one using the PCR machine at the moment, so I can't say If it's working with someone else. But I tried to run the PCR in an older machine (that we dont use anymore) and It didn't work.
A clean gel is good.( but not ideal) It rules out primer dimer or product contamination which can both be a serious problem and also proves you can see product so it is not that the pcr works but you forgot to add ETBR in which case you would see nothing.
Is your primer initially dissolved in water in which case over time depurination can take place with the co2 from the air and the primer may be degraded. If so order new primer and make up in TE for greater stability.
Run another different primer set pcr on a sample that works in pcr to test the pcr machine and reagents are working. If so then definitely order a new primer. Try a pcr at Ta 4c lower than your normal and with 4x as much primer from your freezer stock on a sample that has worked before. If this works then you almost certainly have primer degradation.
Check also that nobody has changed your cycle parameters while you were away...in particular cycle number and Ta. Think positive ….your picture proves the assay works well so it will do again.
Have you noticed if the pcr rection takes longer to complete than usual...this may indicate the machine is struggling to either heat or cool efficiently. Many pcr machines are heated and cooled by peltier junctions and each block may have 3 or 4 of these. If you always put your tubes in the same place on the block then move them to any other position ( preferably not at the edges) in case one junction has failed
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