Jie:LabNotes/ASE/2008-9-22

From ZhangLabWiki
Jump to navigation Jump to search

padlock capture of sample from Yu Gao with eSNP84K[edit]

capture with the padlock probe made on 08-03-27.

 tube 1: G2 (~0.25ug/ul)
 tube 2: 2B (~3.5ng/ul)
 tube 3: 3B (~3.5ng/ul)
 tube 4: H1 (~5ng/ul)
 tube 5: H2 (~100ng/ul)
 tube 6: Jurkat DNA (5ng/ul)
 tube 7: negative control
 
 reaction system                 tube 1          x7 
 H2O                              6.3ul        44.1ul
 10x Ampligase Buffer               1ul           7ul
 probe(2008-03-27,30ng/ul)          3ul          21ul
 template                           2ul          2ul each
                                                                    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    
                                                              x20      4ul           2ul         2ul         4ul     8ul

Reaction program:

  95c 10min -> 63C 14h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.8nM 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.

PCR[edit]

                                                   x7
   Template                            10ul      
   2X iProof Mastermix                 50ul       350ul
   Amp_F2_sol(10uM)(with tail)          4ul        28ul           
   Amp_R_sol (10uM)(with tail)          4ul        28ul
   50X SYBG I                         0.8ul       5.6ul
   H2O                                31.2ul     218.4ul
   

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

File:20080924 Yuan Gao sample SNP84k capture.jpg20080924_Yuan Gao sample SNP84k capture

Qiaquick column purification. Yields:

 G2: 26.1ng/ul (132nM) x 30ul
 2B: 26.8ng/ul (140nM) x 30ul
 3B: 40.7ng/ul (213nM) x 30ul
 H1: 33.4ng/ul (175nM) x 30ul
 H2: 43.3ng/ul (227nM) x 30ul
 Sent 15ul products to Yuan Gao on 09/24/08.