I get bands but unfortunately when I do PCR again to cut the bands and clone, I can't get bands again. I tried too many ways like different cycles, from 30---40. Also checked different temperatures, also different diluted cDNA but can't get result. Any suggestion or guides to help me please? Here also pictures of my PCR attached. In 1st one I get bands but continuously I can't get bands if I do PCR again?
Hi Saad,
Do you still have the 1st PCR samples? Running a repeat PCR on these samples should work.
If they don't then checking that your components are still good would be a good idea. I don't see any positive controls on your gels, so you can't be certain that there's not a problem with one or more of them.
The 1st PCR is not great, have you checked that your target DNA is still OK? Repreping that might be a good thing to try.
-Richard
Hi,
How much you loaded in the first gel? 5 µl? size? is not a good PCR but you only have one band, so you can reamplify this one with the same primers or design a nested PCR with a pair of inner primers...
Good luck
-Alfonso
I see that you have a ladder running alongside your samples, but what are the sizes of those bands? If you don't know the exact size and sequence of the product from your flanking PCR is there any kind of positive control that you can use to help guide your results? Is there a negative control in this picture to make sure you're not getting contamination? Do you have any software to help you estimate the amount of product you have produced?
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