Alice:LabNotes/2008-11-11

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CES27k9bp library prep[edit]

PCR[edit]

  PCR system 1:                                  x96 
  Template (CES27K9bp):                0.1ul    9.6          
  1.1X Platinum Taq supermix            90ul   7910 (7 tubes of 1130ul each)   
  50X SYBG I                           0.4ul     39    
  100uM AP1V41U                        0.2ul   19.2    
  100uM AP2V4                          0.2ul   19.2        
  H2O                                   10ul   1703   
  PCR system 2:
  Template (CES27K9bp):                0.1ul     9.6          
  2X master mix                         50ul    4800 (3 tubes + 1050ul)
  50X SYBG I                           0.4ul      39    
  100uM AP1V41U                        0.2ul    19.2    
  100uM AP2V4                          0.2ul    19.2        
  H2O                                   50ul    4800

94C 5min -> (95C 30sec -> 58C 1min -> 72C 1min) x 22 -> 72C 5min -> 15C hold.

Purified one plate with three DNA concentrator-100 columns, eluted with 150ul EB (only use wash buffer once).

when wash buffer is used twice, the % yield is significantly lowered:
2-15.4 ng/ul *150ul      3-9.3ng/ul *150ul           4-13.5ng/ul*225ul           5-15.6ng/ul*150ul

When it is only used for once, the results are different:
1-46.5ng/ul *225ul (260/280=1.87)       6-62.1ng/ul *150ul (260/280=1.9)
7-24.8ng/ul *150ul (260/280=1.7)        8-45.1ng/ul *150ul (260/280=1.9)

Lambda exo digestion[edit]

  • To 150ul of DNA, add 20ul 10X lambda exo buffer, 10ul 10x lambda exo.
  • 37C 2h. (alternatively: 37C 45min -> 75C 15 min)
  • Purified the digestion Qiaquick columns, eluted with 80ul ddH2O, combine the elution.
  • Yield: 105ng/ul x 750ul (all products combined)

Dpn II & USER enzyme digestion[edit]

  • To 80ul of ssDNA, add 5ul USER enzyme, 37C 8hr.
  • Add 10ul 10X DpnII buffer, 5ul 100uM DpnII-V4 guide oligo
  • 94C5min -> 60C 5min -> turn off and wait for 20min -> add 5ul Dpn II -> 37C 2h.
  • Add 5ul USER enzyme -> 37C 2h -> 75C 20min
  • (alternatively, all the above methods can be done for 37C 1 hr, the 2D gel result looked same for both methods)
  • PAGE purification in 6% TB-Urea 2D gels.
  • DNA were eluted from the shearing gel assembly and Nanosep columns.
  • Ethanol purification and PAGE gel quantification.
  • Yield: 10ng/ul x 160ul
2D gel result:
File:ZhangLab 2 2008-11-16 16hr 59min.jpg   
The gel on the right is the combination of all the product with low %yield after DNA concentrator step.
File:ZhangLab 2 2008-11-16 22hr 14min.jpg 
File:ZhangLab 2 2008-11-16 22hr 46min.jpg