Hi all, I am now working with ITS2 sequencing library preparation. I had observed that after introducing adapters, linkers and barcodes to a primer, the indexed primers have a much lower amplification efficiency and are more prone to form primer dimers. Would it help if I reduce my annealing temperature? Or do you have other suggestions? Many thanks!
Hi,
Adapters or other patches attached to the primers change annealing temperature. I recommend you to optimize your PCR by gradient PCR. A touchdown protocol is also useful.
Cheers
Dear Zixuan Xie,
What Kaan Hurkan wrote is of course true, and therefore prior to using a set of barcoding primers you should i) check bioinformatically if there is high chance of unwanted structures formation (e.g. IDT Oligoanalyzer is good for the job, but it's online only, so hard to use on hundreds of sequences, but for a few tens its fine), the sequences predicted to form secondary structures should be excluded from the set, and ii) the 'good' ones should be checked in PCR and only those pairs displaying high efficiency should be used. In my case, I screened 120 10 nt barcode sequences and only half of them were found to work well with M13/M13R and ITS1/ITS4 seqs.
Best regards,
Marcin Golebiewski
if the problem is reducing primer dimer then 2 things are worth trying. Use less primer. Use a hot start polymerase so that the primers are always in a high temperature environment when they are with the polymerase so may not be able to anneal to each other
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