Pooling samples for library prep using True seq

[vl80]

We have used barcoded primers for a PCR and our aim is to pool 8 samples together before subjecting to library prep using True seq (a modified protocol for amplicons) so that we have 8 samples per index.
Can someone tell me what amount of DNA should be pooled from each sample/
The recommendation I know is conc > 50 ng/microl and amount > 10microg per index. So, for pooling per index, does each sample need to have >10 microg per it or the 10 microg can be made up from the different samples at equal amounts (say 2 microg each).

Thanks a lot

[pmiguel]

We have used barcoded primers for a PCR and our aim is to pool 8 samples together before subjecting to library prep using True seq (a modified protocol for amplicons) so that we have 8 samples per index.
Can someone tell me what amount of DNA should be pooled from each sample/
The recommendation I know is conc > 50 ng/microl and amount > 10microg per index. So, for pooling per index, does each sample need to have >10 microg per it or the 10 microg can be made up from the different samples at equal amounts (say 2 microg each).

Thanks a lot

What "modified protocol"? How are we supposed to advise you on parameters for your protocol that we don't have?

--
Phillip

[vl80]

Sorry, about it. Modification is just skipping the fragmentation but following the other steps of Truseq.

[pmiguel]

So you are ligating Y-adapters onto PCR products? I guess as long as your are coming into the ligation with at least 100 ng of DNA total (that is nanograms, not micrograms) you will probably be fine.

Not sure where you get the >10 ug/index figure. TruSeq DNA libs want 1 ug, not 10.

--
Phillip

[vl80]

Thank you very much. Yes, I read that in Trueseq the requirement is for 1ug. The tests would be done for us for the first time at a commercial venture. Thier requirement states that the conc of DNA should be > 50ng/ul and the total amount to be > 10ug :/

[kcchan]

This is something you should talk to your service provider about. The 10ug spec is the Illumina standard for their official service providers; it's only intended for genomic DNA. Since your samples are not gDNA, you will need to speak with your provider to find out how much you will need to submit. In your case, you probably don't need to provide very much sample as your libraries will only require adapter ligation. You should also ask them about using a PCR free protocol to eliminate any bias in your pool.

[vl80]

Thank you very much for both suggestions. Would do so.