AlanFung:LabNotes/2015/2015-1-22: Difference between revisions

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*She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
*She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
*I will have to cut the get in the range between 300-350np (Do not of over 400bp) since she cropped the images to show me.
*I will have to cut the get in the range between 300-350np (Do not of over 400bp) since she cropped the images to show me.
*There will probably be high background over 400np, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
*There will probably be high background over 400bp, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
*Use one 5-well TBE gel per one pool of libraries
*Use one 5-well TBE gel per one pool of libraries
*The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
*The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
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http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-12-16#PAGE_size_selection_.26_Sequencing_library_information
http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-12-16#PAGE_size_selection_.26_Sequencing_library_information
== <span style="color:blue">Part I: Pooling libraries based on Noi's concentration </span> ==

Revision as of 04:31, 23 January 2015

Size Selection of LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient

Background

  • Kun thinks the results from the experiment Noi did on Illumina’s plasma samples looks promising.
  • He thinks we should repeat the same experiments on all our cancer and plasma samples.
  • We should have all those WGBS libraries ready, so it would be just a BSPP capturing experiment (we might need to make more probes though).
  • Kun asked Noi to help me identify the reagents and design a capturing experiment on all WGBS libraries
  • Noi said she had already finished the BSPP capture on WGBS libraries and she will need my help to pool the libraries and do size selection
  • Noi will update her wiki so I can follow her work
  • Noi used 600ng of WGBS libraries for capture instead of the 400ng for illumina.
  • She reduced the probe to target ratio from 200:1 to 125:1due to limited amount of probes.
  • She also used MONOD V5A probe set (LMS cluster) and the rest of the protocol are pretty much the same.
  • She quantified the library concentration by PAGE quantification, there seems to be a little high variation between different gels
  • She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
  • I will have to cut the get in the range between 300-350np (Do not of over 400bp) since she cropped the images to show me.
  • There will probably be high background over 400bp, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
  • Use one 5-well TBE gel per one pool of libraries
  • The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
  • Noi already filled in the sequencing spreadsheet
  • For Illumina experiment, Noi suggested Li to only pool sequencing libraries and re-purified with 0.7% AMPure beads to concentrate the libraries and remove the remaining primer dimers.
  • However, for Li libraries, I got low background of a smear over 400bp.

http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-12-16#PAGE_size_selection_.26_Sequencing_library_information

Part I: Pooling libraries based on Noi's concentration