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Bad PCR, is it due to conditions, reagents, or lack of DNA template?


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#1 wheatguy13

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Posted 14 March 2010 - 02:30 PM

I did a PCR today on 300 samples in 96-well plates using a primer pair that has worked beautifully for me in the past.

Once I ran my gels I had poor results ;) Well...not poor but some samples, maybe 25% didn't amplify. And I noticed huge bright bands just below or in my wells of the PAGE gel. I assume this is gDNA, which leads me to my question.

My question: How can you tell if PCR failed(besides the absence of a band...duh!) due to a poor mix, conditions, or lack of DNA. I would like to focus on the DNA template availability. If we add template and the PCR fails due to conditions or poor master mix, we would still expect to see gDNA at the top of the gel??? If we have no gDNA (forgot to add it) there would be no band at the top of the gel flurosed by EtBr. Am I correct on this thinking or will DNA be degraded through the PCR process?

I had 6 sample wells that received no DNA because we keep wells G12 and H12 empty for identification purposes. These wells did not fluroesce or have any bright banding around the well as those with DNA template added.

What you all think?

#2 perneseblue

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Posted 14 March 2010 - 02:41 PM

You may not see gDNA as the amount of DNA maybe rather small. Be careful not to mistaken gDNA with a heavy band that gets stuck in the well, which is a sign of heavy contamination.

For myself, i have a PCR reaction whose condition and primers I know will work on the type of gDNA that I repeatedly extract. I run this test PCR on my DNA samples first. If the DNA sample is good, the reaction will work. If it fails, the DNA sample is bad and needs to be cleaned up. If you have a nanodrop spectrophotometer, you could use that to determine the purity of your sample and whether or not you have any gDNA in your extract.
May your PCR products be long, your protocols short and your boss on holiday




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