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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Bizarre problem with PCR
Posted by: wcladman (IP Hidden, New member, 2)
Date: June 19, 2007 02:59PM
For several months I have had erratic results with PCR, either it works fine or there is no product at all. About 6 weeks ago, I completely stopped getting products (ie. bands on gel). After changing all the PCR components, template, trying a different PCR machine, polymerase, etc, nothing helps. There are other people in this lab using the same pipetters and tips (dedicated to PCR only), polymerase and PCR machine and their PCR is working fine. One colleague made aliquots of her own PCR components (buffer, dNTPs, primers, template) that worked for her but they didn't work for me. I asked another colleague to run a PCR reaction using my components and her own in tandem and both worked. On 2 separate occasions, colleagues have watched me set up the PCR reaction and find nothing wrong. I have worked in labs for over 20 years and my techniques are excellent. Something appears to be inhibiting the PCR reaction. On the gels, the DNA ladder is clear, and you can see the template bands very faintly. I am not adding too much or too little template. I have been following the exact protocol that the others have used.
Has anybody any idea what could be causing this? This situation is getting more and more stressful. Serendipity
Re: Bizarre problem with PCR
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: June 19, 2007 04:05PM
What I can think of from your description is that you may have pipetting errors during PCR reaction setup. It sounds to me not related to reagents/cycler etc but your practice. If a set of reaction components work for other of your colleagues, it should also work for you. Keep trying and best of luck!
Re: Bizarre problem with PCR
Posted by: wcladman (IP Hidden, New member, 2)
Date: June 20, 2007 10:31AM
I am very certain that this problem is not due to pipetting errors. But to be sure, I asked 2 of my colleagues to watch me set up. All of the components were added in the correct volumes and the tips carefully rinsed. I am not excessively rough with any of the ingredients and am sure that the reaction is mixed. I also tried out different methods of mixing, but nothing made any difference. This whole thing defies logic and has been going on for several weeks now.
It is as if some sort of inhibitor is getting into the tubes as I am setting them up. We use a dedicated bench, pipettors and filtered tips for PCR. Something prevents the reaction from working.
Re: Bizarre problem with PCR
Posted by: riyaz (IP Hidden, New member, 3)
Date: July 14, 2007 02:28PM
Do not worry everything will be fine, i beleive that there is no pipetting error.
I think you work on your primers. Sometime primer show decrease activity. whtever conc. u r using right now , increase it 4 to 5 fold and use DMSO or DTT and CES , in your PCR reaction. Try to optimise on which primer conc. u will get good bands. Mean time order for new primers also. Hope this will solve your problem. Your can write me to riyazpandit@yahoo.com
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