Jie:LabNotes/ExonomeSeq/2009-1-19

From ZhangLabWiki
Revision as of 01:23, 21 January 2009 by >Jie deng (New page: ==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 ...)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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)