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Hi to all who are currently doing the library prep for GBS
As soon as my adapter oligos arrive they will be resuspended (is this right: if HPSF purified they should be resuspended in low TE (10mM Tris, 0.1mM EDTA, pH 7.5-8.0); if desalted they should be resuspended in Nuclease free water?).
Once resuspended (200uM) there will be a titration to determine the best concentration of adapter with the a single rep of the plant DNA population (is one DNA sample and set of adapters sufficient for the titration or is there a % of samples and/or adapters that need to be assayed?).
Once Bioanalyzer results are available the concentration of adapter is chosen where the library looks good and the primer peak is very low. Is there anything else needed aside from doing the titration for normalizing the ds adapters properly?
Likely to have more postings as I work through this process, and thank you for any input you can provide.
As soon as my adapter oligos arrive they will be resuspended (is this right: if HPSF purified they should be resuspended in low TE (10mM Tris, 0.1mM EDTA, pH 7.5-8.0); if desalted they should be resuspended in Nuclease free water?).
Once resuspended (200uM) there will be a titration to determine the best concentration of adapter with the a single rep of the plant DNA population (is one DNA sample and set of adapters sufficient for the titration or is there a % of samples and/or adapters that need to be assayed?).
Once Bioanalyzer results are available the concentration of adapter is chosen where the library looks good and the primer peak is very low. Is there anything else needed aside from doing the titration for normalizing the ds adapters properly?
Likely to have more postings as I work through this process, and thank you for any input you can provide.