I have increased the extension time as well as the annealing temperature, but still the band is coming above the dye at the bottom. Can anyone help me troubleshoot this problem?
You didn't mention on the size of your PCR product, if your amplicon size is
You didn't mention on the size of your PCR product, if your amplicon size is
First, as our collegue Dhatchana said, which is the size of your PCR product? I think that the DNA fragments you are seeing are dimers of the primers. Please, provide information about the PCR conditions and I will try to help :-)
U didnot mention the amplicon size.... may be optimization condition is not proper ..... u just check the amplicon size and % of gel..... may be primer dimer formed but still need to check ur amplicon size and correct gel % ...
I agree with previous comments that you need to mention the expected amplicon size. If your amplicon size is less than 100bp which is rarely the case, you can use a loading dye containing xylene cyanol. we will be more than happy to help if you can provide further details
Have you run a molecular marker along with your amplicon for comparison? If you amplicon traverses farther than ladder then probably its a primer dimer or a mispriming product.
Another dye alternative that I routinely used was Orange G in 50% glycerol. As the others here have suggested, you need to be more specific in your question in order for us to answer with more clarity.
hey, if you are not getting your PCR product of right size , then your primer might get dimerized in this situation, either use 1-5% DMSO or formamide that will reduce the primer dimer formation. try it once, will surely get your PCR of right size. may be you can use 2-5% glycerol in your PCR reaction mix.
Intersting. Due to the lack of information, I don't see why you wanted to increase extension time. As everybody above have already pointed out, you have to know what size of a PCR product you are expecting. Depending on your application with the PCR product, primer dimers might not matter at all.
Hi!,
As everybody commented first be clear in asking the question with all details so that you get valuable answer for your problem. For this kind of things uploading gel image give clear idea to others. What dye are you using, travelling of dye based on the percentage of gel.
What are you amplifying, what is the size of your product, why did you increase annealing as well as extension temp. No head and tail for you question.
for eg. BPB travells along with DNA fragment of approximately 500 base pairs, in 2% gel
xylene cyanol typically migrates at about the same rate as a 4000 base pair DNA fragment in 1% agarose gels.
So frame your question correctly and get correct answer.
no amplification, using low agarose concentration for gel. I can think of too many variables which can cause this
If the band size is 1.5 kb you will always get it before the BPB dye front in a 1 or 1.% % agaarose gel
your PCR amplification is not the amplified gene
it may be primer dimers, check on ur PCR reaction again
As suggested please make sure that the band below the dyefront is not due to primer dimer
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