Dear Community, I extracted DNA from yeasts and performed a PCR with ITS primers. The PCR bands are not intense and do not allow sequencing. I've tried to extract DNA with a lithium protocol, but the bands are still weak. Do you have any recommendations to have better yields of my PCR products?
do you get better pcr yields if you run more cycles?
Is your sequence GC rich in which case try adding a final concentration of betaine of 1Molar.
You may have pcr inhibitors in your dna. Try running the pcr with1/2, 1/4 and 1/8th the amount of dna and see if the pcr yield increases. Sometimes less inhibition means more amplification so you can get better pcrs with less dna
The sequence is (ITS 4:TCC TCC GCT TAT TGA TAT GC/ITS 1: TCC GTA GGT GAA CCT GCG G) and works very well with a positive control which is a fungus (good bands).
I'll try to increase the cycles and dilute, in fact, we may have inhibitors since during the extraction (without a kit) it is too difficult to separate the phases after adding the phenol-chloroform.
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