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PCR is inconsistent

PCR

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#1 waterwind

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Posted 04 February 2018 - 11:07 AM

Hi everyone, I need your help regarding my pcr problem. So early last week I created a serial dilution of a purified linearized plasmid (around 4 kb). I performed a gradient pcr on one of the dilution solution to determine the optimize annealing temperature. All was done and well.

Next, I did the pcr with the optimized Ta on all of the serial dilution. There was also no problem. I got much PCR product on every samples. On the next day, I wanted to reproduce the result. But this time, there was only smearing in the agarose with the exception of the highest concentrated solution. I tried the pcr again but the weird thing was that the highest concenctrated solution did not show any more band but the second highest one did. The third try: band on the highest and the second highest but still no product on the rest of the samples. How could it happen?? I aliquoted the diluted samples and stored it at -80C, so it shouldn't be degraded that fast!

I'm now running a positive control to test the dNTP, buffer, and polymerase. But if the positive control work but the samples don't, what do you suggest is happening? Is it possible that the primers are degraded? We store the stock solution at -80C and the working solution was freshly made (stored at -20C).

 

I need this PCR to work before thursday... so any help/idea is appreciated. Thank you unsure.png



#2 Mad Researcher

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Posted 07 February 2018 - 05:25 AM

I wouldn't free thaw my DNA samples from that high temperature. Ideally, if you want to use your samples the next day, you should store them at 4°C (for 2-3 days). I have used this method with success. If I want to store it for a month or so, I keep it at -20°C. And if I want to keep it for months or years, I store them at -20°C. 

 

If the +ve control works, then your samples have degraded. Again, I store my working primer stock at 4°C for a week. I make enough to last for a week and I store a batch of working concentration at -20°C. This way, I avoid repeated freeze thaw of the stock. 

 

Did you try measuring the DNA samples on a nanodrop? Do the values vary from the original? How is the A260/230 and A280/230 values?

 

Hope this helps.


Cheers,

Mad Researcher





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