Problem in library enrichment

[meganathan.pr]

Hi,
I got a problem in my library preparations.
I tried to prepare 180/230 bp libraries. I have used NEB reagents and illumina PE adapters and primers for my library preparations.
I am getting the expected size fragments after PCR enrichment. However, the concentration is too low, ~10 ng/ul. I have repeated the same many times but did not get a high yield.
Could anyone please tell me what should I change to get high yield?

Thanks.

[NextGenSeq]

10 ng/ul is enough to sequence. When you dilute a library to 10 nM it is typically about 2 ng/ul

[pmiguel]

And the new, v3, cluster kits offer an option of using 2 nM libraries. That should be available on HiSeqs and HiScanSQs. (Not sure about GAs).

--
Phillip

[meganathan.pr]

Thanks for your reply.
As a beginner, I would like to know your experiences. Is it not looking odd to get such a low amount of amplification when I am using 5 ug of gDNA for my library preparations? or is it normal to get such a low amplification?

[pmiguel]

Yes, but there are lots of things that can go wrong.

First, my experience is that most gDNA concentrations estimation are done by UV spec, which tends to mean that they are a 10-100x overestimate of the true amount of DNA. This is because most genomic DNA preps are largely RNA (possibly degraded with RNAse--but that has little effect on the amount of UV the RNA absorbs.)

So there is that. Past that, the biggest issue is that contaminants in the DNA prep (ethanol, etc.) can inhibit end-repair post-shearing. That can drastically lower yields.

--
Phillip

[sehrrot]

though you got lower than 2nM, you would change conditions for template preparation, which should end up at 12 pM-ish. So, it would not be a problem, but you may concern that your library would have lower complexity.

[Liting]

though you got lower than 2nM, you would change conditions for template preparation, which should end up at 12 pM-ish. So, it would not be a problem, but you may concern that your library would have lower complexity.

Hello，sehrrot.I also have a problem about the PCR richment during library preparation。I also have got some unexpected fragments which are about 100bp smaller than the expected fragments after PCR。 Have you got the same problem？What should I do to avoid the problem？Thank you very much！