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Old 09-24-2010, 06:58 AM   #1
TonyBrooks
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Default Help with denaturing low concentration Illumina PE Library

Hi all
I have an Illumina PE library that is of very low concentration (~1.5nM). If I speedvac I should be able to get 2nM to do the denaturation, but I'll have to do it in 10ÁL rather than the stated 20ÁL as I don't have the volume. Has anyone done this before?
Thanks
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Old 09-24-2010, 09:23 AM   #2
GW_OK
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Add 13.3 ul of your 1.5nM library to 4.6 ul of EB buffer (10mM Tris)
Add 1 ul 2N NaOH and incubate 5 minutes
Add 1 ul 2N HCL to neutralize your NaOH
You've now got 20 ul of a 1000pM ssDNA library.
Dilute to your favorite loading concentration with chilled hyb buffer.

Using this method I have successfully loaded libraries as low as to 193pM.
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Old 09-26-2010, 02:49 PM   #3
ScottC
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I can vouch for that method too... I've successfully sequenced libraries the same way. I think there's another thread on the topic here somwhere.

GW_OK: Out of interest, how do you assess that your low-concentration libraries have been successfully prepared (other than sequencing)? Do you use real-time PCR?

Scott.
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Old 09-27-2010, 08:30 AM   #4
GW_OK
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qPCR with the Kapa kit. It's the only way to go in my opinion. (Well, at least the qPCR part. You can pick your own reagents.)
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