Jie:LabNotes/ExonomeSeq/2009-1-19

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capture of Jurkat with long cosmic probe and ds short cosmic probe[edit]

 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 reaction:

 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.

SLN mix:

                                                                ddH2O   Ligase Buffer   Ligase    Amplitaq    dNTP           
2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.58ul    0.1ul       0.1ul       0.2ul  (10mM)0.02ul 


PCR[edit]

                                      x8
   Template                10ul      
   2X iProof Mastermix     50ul      400ul
   AmpF6.2SoL (10uM)        2ul      16ul
   AmpR6.2SoL (10uM)        2ul      16ul   
   50X SYBG I             0.4ul      3.2ul
   H2O                   35.6ul     284.8ul
   

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 13 -> 72C 5 min -> 15C hold.

 Short Cosmic 3760 had only 17cycles while long probe had 21 cycles.

File:20090122 long and ds cosmic capture.jpglong and ds cosmic capture

 Qiaquick column purification:
 short probe: 15.3ng/ul (36nM) x 30ul;
 long probe (low con.): 32.9ng/ul;
 long probe (high con.): 18.7ng/ul (30nM) x 30ul;
 Sent 15ul short probe captured amplicon (36nM) and long probe (high con.) captured amplicon (30nM) to Yuan Gao.