when I run the gel, GAPDH bands and my desire band stick together.Does anyone knows whats the problem?
what sizes are your amplified fragments please...the percentage agarose gel will have an effect on separation of different size fragments but if the sizes are too similar then agarose will not separate them if both are large fragments. You need to get rid of the primer dimer in the first samples then you will get more product . Also I think that you should run the gel at lower voltage as the samples are smiling so size separation will suffer. Smiling can also be caused by too much salt in the sample so sometimes using less loading dye can have an effect on this. Can you tell us what samples are on the picture please?
Hi. Thanks for your advice.Its a 2% agaros gel and the voltage was 70. The GAPDH band size is 750bp and my gene of interest is 500bp.I didnt use loading dye because my master mix is colored. In the picture the first 4 bands close to the ladder, are the SNP
rs 9935238 of the CYBA gene and the last 4 are the same sample plus GAPDH primers.
ok I am taking the bright band on your size marker as 500bp . Mixing the primers has had 2 effects. First the primer dimer has disappeared and the 500 band is stronger, Second there is no 750 band at all. You need to check that both Gap primers can work at your annealing temperature, I would change to a 1% gel but that is not important. I think you should run an amplification to check primers so if I call your primres af and at for arms and gf and gr for Gap then run a pcr at your current cycling conditions with sample s and primers af+ar, gf+gr, af+gr and gf+ar and also all 4 primers. It is possible that you are getting a wrong product of about the right size from a primer mismatch and this might give a clue but I think that something is stopping the gap primers from working which also seems to improve your arms amplification. It could be impurities in the dna are inhibiting amplification and adding the second pair of primers improves amplification but you still have to account for why Gap is not working. I think this is a pcr problem not a gel separation problem and the 2 most likely reasons may be temperature too high so no annealing of gap primer or that your gap primers have been stored in water or thawed too many times and that the gap primer/primers are degrade
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