Jie:LabNotes/Protocol/exon capture system

From ZhangLabWiki
Revision as of 22:41, 17 October 2008 by >Jie deng
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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.