Hi everyone
I'm trying to clone a gene sequence. I have designed specific primers on the gene sequence, but the PCR has more and more bands. I tried to clone, and later did a PCR with both sequence-specific primers and M13 universal primers, obtaining more bands on the gel. How do I get my one band? Do I have to perform the PCR? What do you suggest? Has anyone had this problem before?
Run your PCR product on a gel and cut out the band you want before doing a gel extraction. Check your primer and template sequence for repetitive sequences and palindromic sequences (did you add a few base pairs 5' of restriction sites on the primers).
Hi Maria Dellino
I agree with Robert Adolf Brinzer
Moreover, you can use additives like DMSO and Betaine and may increase annealing temperature of your PCR to get a single specific band. But to me, it looks like a primer design issue.
Hope it helps,
Subham.
Hi Maria,
I agree with Robert and Subhan, it most likely a problem with primer design.
Maybe you have contamination within the starting sample. Do you have a negative control? Does it also have bands?
First I will make sure that the primers are not the problem: check that they don't bind anywhere else within the template DNA, also if you can design them so that they have a couple of C or G at their 3' end that will increase the affinity to the sequence, make sure that they are not over 5degrees Tm different. And that you are adding the correct primer molar concentration to the reaction!
If you are happy with how you designed your primers and there is no contamination in your starting sample then you should optimize the PCR conditions. You might be seeing multiple bands due to the primers annealing non-specifically to the template, this is normally solved by increasing the annealing temperature. Perform a gradient PCR increasing the annealing temperature in sets of 1 or 2 degrees for each sample, run them on a gel, and see which one has less non-specific bands. If you still have problems there are many troubleshooting guides, I like this one "https://www.bio-rad.com/en-uk/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S"
Good luck!
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