I am trying to optimise my PCR, I know the Fungus is Aspergillus brasilliensis. I am using ITS1F and ITS4 primers with Tm of 56.6 and 61.5 respectively. I have changed cycles, annealing times and temp, extension times. I've done gradient PCR going from 55-60C, then narrowed it to 55-56C and I still get the second band.
Denaturation is 96C for 15 mins
then 94C for 1 min to 30 secs
Annealing has been from 50-60C, from 1 minute to 20 secs
extention from 1 minute to 45 secs at 72C
cycles 30, 35 and 40
Any ideas as every combination I try has this second band
I do not know the distribution of these regions on the genome.. but do you expect to have different band sizes? if there are more ITS copies on the genome this could be a kind of "physiological" if the two regions have different size. Looking at the band intensities it seems not the case, it is most probably an artefact.
If you are not able to correct it playing with temperature, I suggest you extending the electrophoresis time and cut out the bands, extract the DNA for cloning/Sanger sequencing or NGS depending on your experimental design.
bests
Giuseppe
Sorry, when I say artiact I was referring to a strange PCR product coming from a well described kind of quimeric amplicon. Diluting is right. It is also described as overload of DNA compromise the PCR. Try 1:10 and also 1:100.
I hope it works
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