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PCR didn't work


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#1 claritylight

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Posted 01 October 2010 - 06:45 AM

Recently several reactions of my PCR failed to amplify any product. Reading the DNA concentration in the PCR product gave the same concentration of DNA as that before amplification.

I suspect it's my enzyme that I used. My PCR controls also were low concentration, but I used these same controls several times recently in another setup, so I just don't think I got any amplification and I suspect enzyme went bad or it just didn't catalyze the reaction.

Is it possible if I could just take 5-10ul of these PCR product, add in fresh enzyme and repeat thermocycle? Would this work? I don't have enough of my original DNA volume to repeat the entire PCR process over again, so I am trying to work from the PCR products now.

#2 Trof

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Posted 01 October 2010 - 07:46 AM

Did you measure your PCR product before or after you purified it? Primers and dNTPs absorb at same wavelength as PCR products they create, so if you measured it before reaction and after without purifying first, you would see no difference.

If you purified your sample and you're sure there is no product, then I guess it's better to use all your purified volume as a new template (so add everything else, buffer, dNTPs, polymerase, primers..) as it contains a bit less of the DNA than you originaly put into first reaction.

Anyway it's better to visualise it on gel electrophoresis where you can see both amount of product and specificity of your reaction but it has a limit of about 10ng, that's still visible on the EtBr gel. You can see no band on the gel and still be able to get some product if you reamplify it, but in this time use 1 ul or less (dilute it) to the second reaction. You have to decide whether the was no amplification at all, in that case use all the product when you're sure you have working enzyme, or if there was amplification, but not enough to see, then use only 1 ul or less. Or you can try both.

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#3 claritylight

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Posted 01 October 2010 - 09:31 AM

Trof, thanks for the suggestions, I will try them out. They are very helpful




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