I did the PCR and I got the proper bands but next day, I did the same thing but there seems no band after the electrophoresis. I am confused what to do next to get the proper results? Please give me some suggestions.
naturally your first thought is that you forgot to add one reagent but a couple of other possibilities are:
1 Someone changed the annealing temperature on the pcr machine.
2 Your DNA is poor quality and contains pcr inhibitors and you are just on the edge of the pcr working and the second assay you pipetted a small amount more dna and it stopped the reaction. Try a pcr with dna amounts 2x, 1x, 0.5x and 0.25x the amount of DNA that you used in the first assay. Do you know the OD260/280 ratio of your DNA?. Include in this assay a dna that has worked in any pcr previously as a positive control as well as your unknown sample dilutions
3 Set the annealing temperature 2C lower than you used just in case you are just on the edge of primers annealing/not annealing.
4 use the same pcr machine as for the first assay. Older pcr machines do vary in the precision of their temperature control.
5 Was there a low molecular weight bright smear in the sample track....too much primer dimer ( often caused by using too much primer) will inhibit the pcr reaction. It can be minimised by setting up the pcr on ice,using less primer or using a hot start enzyme.
Just repeat again carefully. There is nothing to get confused or ask on researchgate why your single PCR did not work which was working the previous day.
May be there was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don't exceed 50 ng/band. The DNA may be degraded. Avoid nuclease contamination Sonu Singhal
- Badges
- Science method