Tweet
Are you trying to go lower then 100 pM?
-
-
Hi
The concentration is 150pM.
Thanks.Comment
-
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
-
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
-
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!!
Originally posted by acockburn View PostComment
-
During the COVID-19 pandemic, scientists observed that while some individuals experienced severe illness when infected with SARS-CoV-2, others were barely affected. These disparities left researchers and clinicians wondering what causes the wide variations in response to viral infections and what role genetics plays.
Jean-Laurent Casanova, M.D., Ph.D., Professor at Rockefeller University, is a leading expert in this crossover between genetics and infectious...09-09-2024, 10:59 AM -
The first FDA-approved CRISPR-based therapy marked the transition of therapeutic gene editing from a dream to reality1. CRISPR technologies have streamlined gene editing, and CRISPR screens have become an important approach for identifying genes involved in disease processes2. This technique introduces targeted mutations across numerous genes, enabling large-scale identification of gene functions, interactions, and pathways3. Identifying the full range...08-27-2024, 04:44 AM
Comment