Jie:LabNotes/ExonomeSeq/2009-1-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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 ...)
 
>Jie deng
No edit summary
 
(7 intermediate revisions by 3 users not shown)
Line 1: Line 1:
==capture of Jurkat with long cosmic probe and ds short cosmic probe==
==capture of Jurkat with long cosmic probe and ds short cosmic probe==


   tube 1: short cosmic3760 (100ng/ul)
   tube 1: short cosmic3760 (19.3ng/ul,4/29/08. requantify today: 12ng/ul)
   tube 2: long cosmic 3760 (100ng/ul)
   tube 2: long cosmic 3760 (14ng/ul)
   tube : long cosmic 3760
   tube 3: long cosmic 3760 (14ng/ul)
   tube : ds short comic 3760  
   tube 4: ds short comic 3760 (10ng/ul)
   tube 3: ds short cosmic 3760 (200ng/ul)
   tube 5: ds short cosmic 3760 (10ng/ul)
   tube : negative control for short probe
   tube 6: negative control for short probe
   tube 4: negative control for long probe
   tube 7: negative control for long probe
   tube 5: negative control for ds short probe
   tube 8: negative control for ds short probe
    
    
Capture system:
Capture reaction:


   reaction system                             tube      x6                                              
   reaction system         1    2    3    4    5  6  7  8                                              
   H2O                                         5ul      30ul                                     
   H2O                     6    6.2  4.4  4    2  7  7.2 5                                   
   10x Ampligase Buffer                         1ul      6ul                                      
   10x Ampligase Buffer     1 each                                      
   probe(from Alice, 2008-07-06,12ng/ul,370nM) 2ul      12ul
   probe                   2    1.8  3.6 4    6  2  1.8 4
   template (100ng/ul)                         2ul      2ul each
   Jurkat gDNA(200ng/ul)   1    1    1    1    1  0  0  0


Reaction program:  
Reaction program:  
    
    
   95c 10min -> 63C 14h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 60C 4h -> <BR>12 cycels (95C 1min -> 0.1C/sec to 60C, 60C for 4h 55C 1h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
   95c 10min -> 63C 14h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 60C 4h -> <BR>12 cycels (95C 1min -> 0.1C/sec to 60C, 60C for 4h 55C 1h) -> 95C 1min -> 37C 1min -> <BR>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==
==PCR==


                                       x4
                                       x8
     Template                 5ul      
     Template               10ul      
     2X iProof Mastermix    25ul     100ul
     2X iProof Mastermix    50ul     400ul
     AmpF6.2SoL (10uM)        2ul       8ul
     AmpF6.2SoL (10uM)        2ul     16ul
     AmpR6.2SoL (10uM)        2ul       8ul    
     AmpR6.2SoL (10uM)        2ul     16ul    
     50X SYBG I            0.4ul      1.6ul
     50X SYBG I            0.4ul      3.2ul
     H2O                  15.6ul    62.4ul
     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.(Cosmic 3760 had only 17cycles totally)
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.
 
[[Image:20090122_long and ds cosmic capture.jpg]]long 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.

Latest revision as of 18:43, 26 January 2009

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.