Jie:LabNotes/ExonomeSeq/2009-1-19

From ZhangLabWiki
Revision as of 02:05, 21 January 2009 by >Jie deng
Jump to navigation Jump to search

capture of Jurkat with long cosmic probe and ds short cosmic probe

 tube 1: short cosmic3760 (19.3ng/ul,4/29/08. requantify today: 12ng/ul)
 tube 2: long cosmic 3760 (14ng/ul)
 tube 3: long cosmic 3760 (14ng/ul)
 tube 4: ds short comic 3760 (10ng/ul)
 tube 5: ds short cosmic 3760 (10ng/ul)
 tube 6: negative control for short probe
 tube 7: negative control for long probe
 tube 8: negative control for ds short probe
 

Capture system:

 reaction system          1     2    3    4    5   6   7   8                                             
 H2O                      6     6.2  4.4  4    2   7   7.2 5                                    
 10x Ampligase Buffer     1 each                                     
 probe                    2     1.8  3.6  4    6   2   1.8 4
 Jurkat gDNA(200ng/ul)    1     1    1    1    1   0   0   0

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)