Hello,
I extracted my mushroom and got 5 ng dna. Qiagen plant mini extraction.
I used ITS1 and ITS4 primer.
However my pcr failed. Why?
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Hello @GUL,
Could you provide more detail? E.g. by what metric has your pcr failed? Failed sequencing run, no gel band, etc.?
Note that this is a software & bioinformatics focused forum, so questions about wet-lab troubleshooting might not be best served here.
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Ohh yes you right.Sorry about that. I just need a help for fungi but I am not correct platform. I run my pcr product into gel however couldn`t see band. I meant I am seeing band but it is same with my ladder.So my primers (ITS1 and ITS4) not amplify. Pcr reagent buffer10x, primers, MgCl2(25mM), dNTPs(2mm)remain is water and Dna polimerase. TM 55C cycle is 35
Hello @GUL,
What do you mean "it is same with my ladder"? Ladders have multiple rungs, so which one does your product line up with? Do you only see primer dimer? Do you have any negative controls to compare to?
Yes. I see only primer dimer.
Hello @GUL,
There are many things that could have gone wrong. Did you ever quantify your mushroom DNA extraction to ensure that that's not the issue? I would double check everything--the primer sequences, their concentrations, the master mix protocol, the PCR protocol, and simply try again. Only do a couple of samples until it's working so you don't waste reagents or template.
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