Jie:LabNotes/ExonomeSeq/2009-1-19
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capture of Jurkat with long cosmic probe and ds short cosmic probe
tube 1: short cosmic3760 (100ng/ul) tube 2: long cosmic 3760 (100ng/ul) tube : long cosmic 3760 tube : ds short comic 3760 tube 3: ds short cosmic 3760 (200ng/ul) tube : negative control for short probe tube 4: negative control for long probe tube 5: negative control for ds short probe
Capture system:
reaction system tube x6 H2O 5ul 30ul 10x Ampligase Buffer 1ul 6ul probe(from Alice, 2008-07-06,12ng/ul,370nM) 2ul 12ul template (100ng/ul) 2ul 2ul each
Reaction program:
95c 10min -> 63C 14h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 60C 4h ->
12 cycels (95C 1min -> 0.1C/sec to 60C, 60C for 4h 55C 1h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
PCR
x4 Template 5ul 2X iProof Mastermix 25ul 100ul AmpF6.2SoL (10uM) 2ul 8ul AmpR6.2SoL (10uM) 2ul 8ul 50X SYBG I 0.4ul 1.6ul H2O 15.6ul 62.4ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 13 -> 72C 5 min -> 15C hold.(Cosmic 3760 had only 17cycles totally)