Kun:LabNotes/CpgSeq/2008-3-18: Difference between revisions
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[[Image:2008-03-21-capured-amplicon-AluI-cut.png|600px]] | [[Image:2008-03-21-capured-amplicon-AluI-cut.png|600px]] | ||
==PAGE purification of amplicon D, F, and Cosmic C(03/17/08)== | ==PAGE purification of amplicon D, F, and Cosmic C(03/17/08)== | ||
Resuspended the pellets in 15ul ddH2O. | [[Image:2008-03-21-PAGE.png|350px]] | ||
Resuspended the pellets in 15ul ddH2O. | |||
==Blunt end cloning== | ==Blunt end cloning== | ||
PAGE purified amplicons: 3.5ul | PAGE purified amplicons: 3.5ul |
Revision as of 02:48, 23 March 2008
Exp. 1 Cpg capture with a new batch of bisulfite treated Jurkat DNA from 03/17/08
Sigma x2 Zymo x 2 Template 7 3 10X buffer(fresh) 1 1 Cpg28k probe(218nM) 2 2 H2O 0 4 Tubes: A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zyno, dNTP 50uM
Exon capture control
GM20431 131ng/ul 7 (10/12/2006) 10X buffer(fresh) 1 E55kv1 probe(220nM) 2 (01/12/2008) H2O 0
94C 10min -> 60C 10min -> add 1ul NSL mix (Stoffel 2U/ul, Ampligase 0.5U/ul, dNTP 10nM or 50uM) -> 60C 1h -> 45 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul Exo I&III, 37C 2h -> 94C 5min -> 4C hold.
PCR
Template 12ul 2X iProof Mastermix 100ul 100uM AmpF6.2 0.8ul 100uM AmpR6.2 0.8ul 50X SYBG I 0.8ul H2O 85ul Split into two tubes. 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 15 -> 72C 5 min -> 15C hold.
File:2008-03-21-Cpg-E55kv1-captured-PCR.png A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zymo, dNTP 50uM E: E55kv1, dNTP 10nM F: E55kv1, dNTP 50uM
To test whether some of the high MW bands are concatemers, I did Alu I digestion on the MinElute purified amplicons. 8ul DNA + 1ul 10X NEBuffer2 + 1ul Alu I -> 37C 30min -> 75C 10min. File:2008-03-21-capured-amplicon-AluI-cut.png
PAGE purification of amplicon D, F, and Cosmic C(03/17/08)
File:2008-03-21-PAGE.png Resuspended the pellets in 15ul ddH2O.
Blunt end cloning
PAGE purified amplicons: 3.5ul 10X ligation buffer: 0.5ul Zero Blunt vector: 0.5ul T4 DNA ligase: 0.5ul 16C 1h -> 4C overnight.