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Old 06-09-2008, 02:10 PM   #1
ScottC
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Default qPCR quantitation

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.
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Old 06-09-2008, 10:03 PM   #2
ECO
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With regard to illumina data, i have also heard people stain it with sybrgreen or similar to check cluster formation before starting a run.
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Old 06-10-2008, 05:33 PM   #3
ScottC
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Quote:
Originally Posted by ECO View Post
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.
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Old 07-08-2008, 06:21 AM   #4
sblake
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Default 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
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Old 07-10-2008, 07:38 AM   #5
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Quote:
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
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Old 07-10-2008, 08:04 AM   #6
sblake
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Default qPCR

Esther - good to hear...any instances it was grossly inaccurate or failed due to difficult template (perhaps making it impractical at times)? Danke
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Old 07-10-2008, 08:28 AM   #7
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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...
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Old 07-10-2008, 08:44 AM   #8
sblake
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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
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Old 07-10-2008, 09:41 AM   #9
elg
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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
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