Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies
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i have done PCR by using new set of primer but there is no result on 2% agarose gel except 100bp ladder.PCR conditions were already optimized as previous primer set was degraded so i use new primer set on same conditions,,what are the reasons of no result?
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That is a very broad question and impossible to give a definite answer to without us being in the lab with you looking over your shoulder as you set it up.
Can you see the primers on the 2% gel? What is your template? Could it be degraded? What is the size of the expected product (as your running a 2% gel I am guessing 200 - 500 bp)? Could it be the Taq? Did you add all of the components ("ticking" them off as you add them)?
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If your primers are dissolved in TE buffer and are kept frozen, there is little chance of degradation.
I guess you have not mixed the PCR mixture and template well or the concentration of new primers is way off.
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Since you didn't solve your PCR problem by buying new primers, what if the old primers weren't degraded, but there was some other problem instead?
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Hi, why dont you nanodrop the primers first, and bring it to working stock (usually 10 or 20 uM). I usually nano drop template as well (DNA/cDNA or RNA in the case of 1 step PCR as opposed to 2 step) and bring it to maximum of 25ng/ul and add 2ul this plenty in case of DNA template. Finally if you are having issues have a PCR control (a reference primer or a known primers that works well on your target template). I recently had issues with my pcr and set up various controls and finally found it was the dNTPs had gone off because of the numerous freeze/thaw cycles.
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