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Are you trying to go lower then 100 pM?
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Hi
The concentration is 150pM.
Thanks.Comment
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Assuming you want to load 10 pM, 1% spike in of PhiX and using the above subnanomolar protocol.
Add the following --- Volume (uL)
0.15 nM sample DNA --- 52
0.2 N NaOH --- 52
Denature 5 min.
Add 20 uL of Tris-HCl pH 7.3 to Hyb buffer.
Add the following --- Volume (uL)
Denatured DNA --- 104
Neutralization buffer --- 208
Add a volume of Tris-Hyb buffer to the desired final loading concentration which is assumed to be 10 pM, so factoring in the PhiX.
Add the following: --- Volume (uL)
Denatured/diluted library --- 311.11
Chilled Hyb buffer --- 458.89
Check pH with indicator paper. It should be between 7 - 8.5.
FINAL LOAD LIBRARY
Add the following: --- Volume (uL) Final Concentrations
10.1010101 pM Denatured Lib --- 693 -- 10 pM
10 pM PhiX Library --- 7 -- 0.1 pM
Load 600 uL into cartridge.Last edited by acockburn; 02-24-2015, 08:27 AM.Comment
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Thank you. What I ended up doing is to combine my low concentration libraries with phix to generate a 2nm library. I do not need lots of reads and merely for verifying sequence. It still failed because of over clustering. I feel as my SybrG assay was not that accurate. I am going to run digital pcr to get more accuracy.Comment
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Neutralization Buffer
Hello,
I came across acockburn's suggestion on using the protocol published in the Supplementary Protocol 12: Modified hybridization buffers of the following paper: A large genome center's improvements to the Illumina sequencing system for sequencing low concentrated libraries.
My question is on preparing the neutralization buffer, it is not clear to me whether I should add the 1 M Tris-HCl pH7.3 in the neutralization buffer or is this only added to the HT1?
Please let me know if anyone has prepared this buffer before.
Thanks a lot!!
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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