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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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reproducibility of PCR
Posted by: Leanne Bedard (IP Hidden, New member, 1)
Date: June 30, 2005 09:43AM
I have been trying to amplify a 600 bp fragment of the 5' flanking region of CYP1A2 and I'm having problems with reproducibility of my PCR. For some samples, the reaction works great, but for others, the PCR product is weak or I get no product. This problem persists even if I use a new batch reagents. I've also tried to optimize the PCR method, but this doesn't seem to improve the PCR for which I get weak products, but makes it worst. And the samples for which I was previously getting a lot of PCR product, I get an even weaker signal or no product at all. I don't think that there is some problem with my DNA samples, since samples that haven't worked previously, will suddenly work (and they have worked for other genes). If every time I run PCR, only 40% of the samples work, it's not very efficient!
Kindly advise
Re: reproducibility of PCR
Posted by: Wangh (IP Hidden, Unregistered user, )
Date: July 1, 2005 07:54AM
hi,
You may want to check the PCR cycler. I had the similar problem before and I found it is caused by the heat block. Some wells in the block don't work and you can check them out with a thermometer. This is just one possibility. If you have ruled out all other possible cause, you should check this out too. Good luck.
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