Hello everyone,
I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for instance. However, I seem to remember not missing any steps in my protocol. One thing that I didn't do, however, was vortex my primer + master mix solution together after adding them to a 1.5 mL tube. I doubt this would cause me to have no bands, but would this really affect my results? Thank you!!
Perform pcr after adding DNA to the working solution u made for pcr.
Reason for no bands is probably first check ur primers are they working? and second is may be you missed DNA sample
Do your primers self-dimerize a lot, or do they form fairly stable hairpin loops?
Hello!
no bands in the gel means absence of EtBr in the gel or some error in the gel components it can be gel itslef or the TAE buffer. Even in the absence of amplification you should be able to see template DNA and primers for sure. there can be no other reason for not seeing dna in the gel. Even if the concentration of primers is low, still they should be visible under UV illuminator.
There can be many reasons why you were unable to see bands in the gel. Here are some which i want to point out:
a) absence of template DNA or the concentration of the template DNA could be very low for amplification.
b) low primer concentration. Check primers once whether they are specific for your template DNA, if it’s rightly made and still not getting amplification, you can try increasing the Primer concentration.
c) Whether the Mastermix is functional. Try keeping a positive control along with your pcr that you want to do.
d) after adding all the components vortex the tube to ensure proper mixing of all the component.
e) try doing gradient pcr, if all the above mentioned points are correct. Maybe you are stuck with the temperatures and the time.
f) check for the EtBr in the gel, in the absence of etbr nothing will be visible on gel.
Andrew Daab From the previous PCRs we've done, no. Using the same reagents, we've had perfectly fine results in the past.
Kanchan Chauhan We had a functional ladder, so it's definitely not related to the gel. Sample-wise, I may have just forgotten to add the template DNA, but I remember distinctly adding the positive control, so I'm not sure.. I'll look into these other reasons, though. Thank you!!
Even in the absence of template, you should be able to see the primers. In the presence of amplification you see some amount of unused primers as the concentration of primers is high (1X of the total pcr mixture) compared to the template which is generally very low.
in the absence of amplification, primers are not used at all so in that can also you should be able to see the primers.
So, according to me try to dissolve fresh primers and try pcr again. if it doesn’t work maybe try increasing the concentration of primers and try pcr again and if this also fails than the primers are problematic.
see in the first well, no amplification occured but template was added. You can see quite alot of primers. In the second well amplification occurred, still alot of unused primers are there. So primers have some issue.
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