qPCR quantitation

[ScottC]

Does anyone here do it to check the efficiency of adapter ligation, and to check the 'real' amount of DNA molecules present for flowing onto the cell?

Cheers,

Scott.

[ECO]

With regard to illumina data, i have also heard people stain it with sybrgreen or similar to check cluster formation before starting a run.

[ScottC]

With regard to illumina data, i have also heard people stain it with sybrgreen or similar to check cluster formation before starting a run.

Yeah, there's a few QC steps that people are using, those two amongst them... I've been trying to get a handle on how many labs use these. I'm not sure if we'll imlement any or all of them.

[sblake]

qPCR quantitation and SYBR cluster detection

Hi there-

Can someone comment on the efficiency of qPCR to do library prep quantitation...I am planning to test this having designed a few primer sets. Is it working accurately for anyone with a specific size selection strategy and for what preps?

(I'm referring to the assumed applicability of this approach to Solexa libraries, from "From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing")

My thought is that if I can establish fairly stringent pcr conditions (maintaining relatively good E) for different sized libraries that have a complex pool of fragment sizes, that next level of quantitation would be nice for us (to avoid titrations w/ new libraries and tweek libraries that start with below recommended amounts of template before sample prep (particularly CHiP-Seq).

Also, if anyone cares to comment in detail about how to perform the so-called SYBR method of evaluating the flowcell clusters that leaves is amenable to subsequent sequencing...that would also be great.

Thanks so much,
Sean

[elg]

I'm referring to the assumed applicability of this approach to Solexa libraries, from "From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing"

so if you follow the protocol as it is described in the paper but with Solexa specific primers it works perfectly.

Cheers
Esther

[sblake]

qPCR

Esther - good to hear...any instances it was grossly inaccurate or failed due to difficult template (perhaps making it impractical at times)? Danke

[elg]

I've done q-pcr with four smallRNA libraries and all four gave a distinct band with the expected size and the quantification was very accurate. So far I can't complain...

[sblake]

that's good, for other applications (gDNA and CHiPSeq) i presume it should be ok if I can keep the size range relevant to pcr and detect when the reaction preferentially amplifies certain fragments. off we go...thanks and cheers

[elg]

yes, you are right with the pcr conditions and should also work with other applications.

BTW, did anybody try to prepare DGE-NlaIII libraries with less starting material than the recommended? and if so, which was the lowest amount that yielded a successful library?

Esther