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Old 10-29-2012, 07:26 AM   #1
theward
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Location: London

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Default Library quantification for pooling question

Hi,

I was wondering if anyone had any success with accurately quantifying libraries for pooling other than using the Qubit? I was wanting to make the process alot more high-throughput. Has anyone successfully used the Agilent Tapestion for this?

Thanks
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Old 11-14-2012, 01:24 PM   #2
rflrob
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Our facility (VCGSL at UC Berkeley) uses the Kapa qPCR kit, and we tend to get really even distributions.
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Old 11-15-2012, 06:27 AM   #3
kwaraska
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Default our Quant

We use a homebrew that is essentially the Kapa kit but using PhiX as the standard for the standard curve.

We found the Kapa kit to be cost prohibitive but are looking into the new Qiagen Quant kit now
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Old 01-17-2013, 11:04 AM   #4
ryclone
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Quote:
Originally Posted by kwaraska View Post
We use a homebrew that is essentially the Kapa kit but using PhiX as the standard for the standard curve.

We found the Kapa kit to be cost prohibitive but are looking into the new Qiagen Quant kit now
Hey kwaraska, our facility also is currently using Kapa for library quantification and while it works great I would very much like to move toward a less expensive alternative. Are there any special tricks to using PhiX as a standard? Would you mind sending me your protocols? Thanks.

ryclone
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Old 01-17-2013, 11:27 AM   #5
kwaraska
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I can send you our "by hand" protocol.

The robotic one isn't as easy to translate into "by hand"

If you give me an email address I'd be happy to share
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Old 01-17-2013, 12:18 PM   #6
ryclone
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Quote:
Originally Posted by kwaraska View Post
I can send you our "by hand" protocol.

The robotic one isn't as easy to translate into "by hand"

If you give me an email address I'd be happy to share
Thank you very much! The email address is wvugenomics@gmail.com
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Old 04-02-2013, 06:33 AM   #7
yun.zhao
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Kapa qPCR kit is good. But I usually qualify each lane's samples by Qubit then pool together then run Qpcr
it save some time and works fine.
Yun
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Old 04-02-2013, 06:57 AM   #8
kwaraska
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Pooling prior to qPCR is fine if you aren't super interested in exact pooling. Most of our customers would prefer to pay a bit extra for additional qPCR rather than take a chance on getting only 10% of one sample when they expect 25%. It is cheaper to pool after qPCR than run another whole lane of sequencing to get more data
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