Replicates of PCR

[daniell]

Today I run 10 replicates of PCR with the same condition and materials but after running gel, only the 1st replicate contains the bands that I want. Whereas the rest only have primer dimer. Any idea? Anybody?

[LilKim]

did you make a master mix? Did you run it all in the same PCR machine? Are some wells of your PCR machine cycling improperly?

[daniell]

I didn’t make a master mix. It was run in the same PCR cycler. I didn’t take into consideration different well will give different result, so don’t know if it is the same well. Other than that, should I change all the reagent for the PCR?

[LilKim]

I hope i’m not making your paranoid about the machine. But, it’s happened to me before… I was doing an rt reaction (i actually dissected about 40 rat brains for my initial mRNA sample). And 30 of my samples worked.

(because half of the block wasn’t cycling properly… )

So, i was PRETTY upset that the PCR machine was "Sick" … i ended up having to dissect a few more brains to re-establish my collection.

anyways.

I think it would be unlikely that your reagents went bad… because if one of your samples worked then the reagents were probably ok. Remember to add taq. last (and keep the samples on ice after you add taq … if it’s going to take you longer that 5 mins to get the samples in the thermocycler).

And… double check that you’re adding everything in each tube. Make a checklist.. or do SOMETHING (.. i’ve been PCR’ing for a while… and you’d be suprised how may times i’ve forgotten to add dntp’s or one of the primers or added the wrong primer)

good luck.
-kim

[daniell]

Thanks Kim for answering.

[daniell]

I use pfu DNA polymerase to run my PCR replicates (which do not come out). After that, I run PCR using Taq polymerase to check if the block of the PCR cycler is doing o.k. I obtain a very bright band. Now, I think the problem is the pfu DNA polymerase! I don’t have other pfu DNA polymerase, therefore cannot confirm my teory. Any idea??

[LilKim]

I think i’d agree with you… Has the pfu polymerase ever worked? Do you think one of your buffer concentrations may be off ?

I’ve never used pfu (only read about it)… but i’d guess that something is wrong with it if it has never worked … or maybe a concentration is off somewhere (check the manufactures packaging stuff .. do whatever the insert says and re-determine all of your concentrations)

good luck?

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