Hi all,
I would like some advice on how to calculate the concentration/dilution of PCR amplicons for next-generation sequencing (NGS) library prep.
I did long-range PCRs for seven candidate genes for 38 human subjects. I need to prepare my samples for the NGS using the Illumina Nextera XT library prep protocol. The Nextera XT Library prep protocol requires 1 ng of DNA (5 ul) as the starting DNA material. I quantified all seven PCR products of each subject on Qubit, and I have varying quantities of DNA for each amplicon. I need to pool all seven PCR amplicons of a subject with equimolar amounts to prepare libraries. The final concentration of the pooled amplicons should be 1 ng (5 ul) as I already mentioned. I think my questions are relatively simple: First, how much of each seven amplicons (in ng/ul) should I add in the pool in order to have equimolar amounts of each amplicon in the 1 ng (5ul) pool? And second, how do I calculate how much Tris-HCl to use to dilute each amplicon in order to reach the required equimolar amounts in the pool?
Thanks in advance!
You can use molecular calculators like this
https://nebiocalculator.neb.com/#!/dilution
you give the length of the amplicon and the desired molarity.
I would choose a small volume that all of the samples can be diluted to. Say 0.5ul. Dilute all of the samples to 1ng/0.5ul using C1V1=C2V2 then mix equal volumes of all samples . With 7 dna samples that mixes to 3.5ul at a concentration of 1ng/3.5ul. Then add water 1.5ul to make up 5ul and that will be 5ul at 1ng/5ul of each amplimer. Scale up as appropriate.
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