Jie:LabNotes/Protocol/exon capture system: Difference between revisions

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>Jie deng
(New page: ==Capture system== the molar ratio of probe/target should be at least 125:1 or 250:1. reaction system H2O...)
 
>Jie deng
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==Capture system==
==Capture system for exon==


   the molar ratio of probe/target should be at least 125:1 or 250:1.
   the molar ratio of probe/target should be at least 125:1 or 250:1.


   reaction system                                                                           
   reaction system                                                                           
   H2O                                          5ul                                        
   H2O                                          5ul  
  10x Ampligase buffer                        1ul                                     
   cosmic probe                                2ul       
   cosmic probe                                2ul       
   template (100ng/ul)                          2ul       
   template (100ng/ul)                          2ul       
Line 10: Line 11:
Reaction program:  
Reaction program:  
    
    
   95c 10min -> 63C 14h -> add 1ul SLN mix -> 60C 4h -> <BR> 8 cycles (95C 1min -> 0.1C/sec to 60C, 60C for 4h), add SLN mix every 12 hours -> 95C 1min -> <BR> when temperature cools down to 37C, add 2ul Exonuclease I/III mix immediately -> 37C 2h -> 94C 5min -> 4C hold.
   95c 10min -> 63C 14h -> add 1ul SLN mix(0.2mM dNTP) -> 60C 4h -> <BR> 8 cycles (95C 1min -> 0.1C/sec to 60C, 60C for 4h), add SLN mix every 12 hours -> 95C 1min -> <BR> when temperature cools down to 37C, add 2ul Exonuclease I/III mix immediately -> 37C 2h -> 94C 5min -> 4C hold.


   SLN mix:
   SLN mix:

Latest revision as of 22:41, 17 October 2008

Capture system for exon[edit]

 the molar ratio of probe/target should be at least 125:1 or 250:1.
 reaction system                                                                           
 H2O                                          5ul    
 10x Ampligase buffer                         1ul                                      
 cosmic probe                                 2ul      
 template (100ng/ul)                          2ul       

Reaction program:

 95c 10min -> 63C 14h -> add 1ul SLN mix(0.2mM dNTP) -> 60C 4h -> 
8 cycles (95C 1min -> 0.1C/sec to 60C, 60C for 4h), add SLN mix every 12 hours -> 95C 1min ->
when temperature cools down to 37C, add 2ul Exonuclease I/III mix immediately -> 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]

   Template                13ul      
   2X iProof Mastermix     50ul      
   AmpF6.2SoL (10uM)        4ul      
   AmpR6.2SoL (10uM)        4ul          
   50X SYBG I             0.8ul      
   H2O                   28.2ul     
   

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