[acockburn]

Are you trying to go lower then 100 pM?

[rpm27]

Hi
The concentration is 150pM.
Thanks.

[acockburn]

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.

[rpm27]

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.

[sriman]

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!!

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.