Alice:LabNotes/2010-1-21: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Zsakura2
>Zsakura2
Line 17: Line 17:
  concnetration(ng/ul): 4.4ng/ul      4.5ng/ul      2.9ng/ul   
  concnetration(ng/ul): 4.4ng/ul      4.5ng/ul      2.9ng/ul   
  volume needed (ul):  5ul          2.4ul        5.5ul       
  volume needed (ul):  5ul          2.4ul        5.5ul       
  --------------------------------------------------------------------------
  -------------------------------------------------------------------
  total (ng):          22ng          11ng          16ng         
  total (ng):          22ng          11ng          16ng         
CViB-ab:
(a)1.5ul + (b)1.5ul
CViB-cde:
(c)1ul + (d)1.6ul + (e)1.5ul


  PCR
  PCR
  Template:         0.25ul
  Template:                   0.25ul
  2x Phusion master mix:   50ul     
  2x Phusion master mix:     50ul     
  100uM AmpF6.3NH2         0.2ul     
  100uM AmpF6.3NH2           0.2ul     
  100uM AmpR6.3NH2       0.2ul     
  100uM AmpR6.3NH2           0.2ul     
  50X SYBG I:             0.4ul     
  50X SYBG I:                 0.4ul     
  H2O                     50ul     
  H2O                         50ul     
  98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold.
  98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold.

Revision as of 22:18, 21 January 2010

MmeI digestion on CViB sequencing libraries

  • Previous library construction protocol can be found under labnotes 1-9-10
  • size selection protocol can found under labnotes 1-15-10
setup:
DNA(15nM)                   10ul
10x buffer 4                2ul
1mM SAM                     2ul
MmeI(2U/ul)                 2ul
37C 1hr, 80C 20min

RE-amplify CViB sets for Covaris shearing

  • previously captured libraries on CViB and PGP7 sets
  • the protocol used for the previous steps can be found under labnotes 12-18-09 and 1-5-10 (for CViB sets) and 8-31-09 (for PGP7)
  • for PGP7, capture products with Jan06 and Jan07 probes were disregarded
     PGP7                   1             2            3          
concnetration(ng/ul): 4.4ng/ul      4.5ng/ul      2.9ng/ul  
volume needed (ul):   5ul           2.4ul         5.5ul       
-------------------------------------------------------------------
total (ng):           22ng          11ng          16ng        
CViB-ab:
(a)1.5ul + (b)1.5ul
CViB-cde:
(c)1ul + (d)1.6ul + (e)1.5ul
PCR
Template:                   0.25ul
2x Phusion master mix:      50ul    
100uM AmpF6.3NH2            0.2ul    
100uM AmpR6.3NH2            0.2ul    
50X SYBG I:                 0.4ul    
H2O                         50ul    
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold.