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

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Line 9: Line 9:
  MmeI(2U/ul)                2ul
  MmeI(2U/ul)                2ul
  37C 1hr, 80C 20min
  37C 1hr, 80C 20min
 
TBE gel result:
[[File:ZhangLab_2 2010-01-23 18hr 01min.jpg]]
==RE-amplify CViB sets for Covaris shearing==
==RE-amplify CViB sets for Covaris shearing==
*previously captured libraries on CViB and PGP7 sets
*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)
*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, therefore only a,b,c subsets were used
*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):  2.5ul        1.2ul        2.75ul           
 
CViB-ab:
(a)1ul + (b)1ul
CViB-cde:
(c)1ul + (d)1.6ul + (e)1.5ul


  PCR
  PCR                                                       x8(for each set)
  Template:         0.25ul
  Template:                   ~0.25-1ul        (total of 2ul for CViB-ab, 4.1ul for CViB-cde, 6.45ul for PGP7)
  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. (11 cycles for PGP7)
Qiaquick column purification and elute in 40ul ddH2O.
Nanodrop:
CViB-ab: 13.7ng/ul
PGP7: 46.7ng/ul
CViB-cde: 58.6ng/ul

Latest revision as of 18:57, 28 January 2010

MmeI digestion on CViB sequencing libraries[edit]

  • 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
TBE gel result:
File:ZhangLab 2 2010-01-23 18hr 01min.jpg

RE-amplify CViB sets for Covaris shearing[edit]

  • 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):   2.5ul         1.2ul         2.75ul            
CViB-ab:
(a)1ul + (b)1ul
CViB-cde:
(c)1ul + (d)1.6ul + (e)1.5ul
PCR                                                        x8(for each set)
Template:                   ~0.25-1ul        (total of 2ul for CViB-ab, 4.1ul for CViB-cde, 6.45ul for PGP7)
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.  (11 cycles for PGP7)
Qiaquick column purification and elute in 40ul ddH2O.
Nanodrop: 
CViB-ab: 13.7ng/ul
PGP7: 46.7ng/ul
CViB-cde: 58.6ng/ul