Hello,
I was doing a PCR for influenza viruses ( more specifically defective interfering segments) and i had good results beside 2 segments. I store them at -20 degrees and i was thinking to run them 5 cycles more ( initially all the sample were run for 15 cycles) . Do i need to make fresh samples or i can just reuse the 2 stored samples. I'm thinking just to place them in the PCR machine and add 5 more cycles to this 2 samples .
Will this work ?
I know the buffer, dNTPs , Taq polymerase and others are limited and have a specific life expectancy of PCR cycles, but 20 ( 15 already done + 5 which i will run )cycles from my presumption will not exhaust all the dNTPs and other reagents.
Thanks a lot ! Sorry for this stupid question !
Difficult to answer with more accuracy given the level of detail provided (what does 'good results' mean?), but...
A) why only 15 cycles? Are you trying to make this semi-quantitative, or just get a yes/no? If the latter, use a full 35 cycles. If the former, consider using qPCR instead?
B) the issue is not exhaustion of PCR reagents, the issue is freeze/thaw damage to your polymerase: most polymerases do not endure repeated freeze/thaw cycles, especially when diluted out to final working concentration. You might defrost your PCRs, put them in the machine, and gain nothing because all your enzymes are denatured.
Maybe consider taking an aliquot of your 15-cycle sample and using it to seed a new reaction?
John Hildyard I know what you mean with the damaging of the polymerase (I know the PCR reaction also influence the degradation level of the regents such as Taq polymerase and dNTPs. ) but beforehand of freezing the eppendorf tube i read this article from the Taq polymerase provider which say :
" At lower temperatures, Taq polymerase is stable for even longer time. Storage at -20 °C is therefore recommended in order to guarantee maximal shelf life. "
And i was just hoping reusing the samples will work in some sort of level.
According to the first question , in this stage of the experiment is just based on yes or no conclusion ,due to low experience in the laboratory, research and more other skills ( I just finish my second year).
Anyway, thanks a lot for your quick response, it helped me to rethink my methodology.
That likely refers to the concentrated stock, usually in 50% glycerol (so it doesn't freeze even at -20): enzymes last longer at cold temperatures, provided they don't actually freeze solid (ice crystals can do bad things to proteins).
Once you've diluted your Taq to make your PCR reaction mix up, it will freeze solid, and this will usually result in a stark drop in viability.
I think it will be difficult to get further polymerization of your pcr product even it stored at -20. I suggest to do the same reaction making freshly.
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