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

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(New page: ==capture system== the molar ratio of probe/target should be at least 125:1 or 250:1. reaction system H2...)
 
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==capture system==  
==capture reaction==  


   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.

Latest revision as of 18:21, 11 June 2009

capture reaction[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(0.8nM dNTP) -> 60C 4h -> 
12 cycels (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:
 
 dilute the 10mM dNTP to 2nM.
                                                                     ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP  
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.8nM dNTP      0.2ul         0.1ul       0.1ul       0.2ul    (2nM)0.4ul    
                                                            

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 8 -> 72C 5 min -> 15C hold.