I'm preparing dual-indexed libraries using NEBNext Ultra Library Prep for targeted resequencing on Illumina MiSeq and can't decide on the number of PCR cycles to use. I started the library preparation with 500ng of input DNA (custom amplicons). After the adapter ligation and bead clean-up I quantified my samples again (only quantified 9 random samples out of all 80) and found concentrations between 180 and 450 ng total DNA.
I'm looking for two pieces of advice. First, what is a good amount of DNA to use in the final enrichment? The protocol says to use it all of the adapter ligated DNA fragments, but I wonder if I could get away with only using a portion, just in case I need to repeat the enrichment step. And my second question is, how should one choose a good number of cycles for the enrichment step?
I have not spoken with NEB about these particular questions yet. However, when I contacted them previously about how much starting amplicon DNA to use, NEB tech support said, "For example if you are using 100ng of amplicons as starting material you may want to try about 4-5 cycles of PCR. I would expect that amplicons may need less cycles of PCR than our recommendations in the manual."
Given that I started with 500 ng of amplicon DNA and don't seem to have lost a great deal of DNA through the preparations so far I'm not sure how many cycles to use. Because my starting material was amplicons I would like to minimize as much additional bias as possible by limiting the number of enrichment cycles.
Thank you for any advice you may have.
I'm looking for two pieces of advice. First, what is a good amount of DNA to use in the final enrichment? The protocol says to use it all of the adapter ligated DNA fragments, but I wonder if I could get away with only using a portion, just in case I need to repeat the enrichment step. And my second question is, how should one choose a good number of cycles for the enrichment step?
I have not spoken with NEB about these particular questions yet. However, when I contacted them previously about how much starting amplicon DNA to use, NEB tech support said, "For example if you are using 100ng of amplicons as starting material you may want to try about 4-5 cycles of PCR. I would expect that amplicons may need less cycles of PCR than our recommendations in the manual."
Given that I started with 500 ng of amplicon DNA and don't seem to have lost a great deal of DNA through the preparations so far I'm not sure how many cycles to use. Because my starting material was amplicons I would like to minimize as much additional bias as possible by limiting the number of enrichment cycles.
Thank you for any advice you may have.
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