Alice:LabNotes/2008-12-15

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Revision as of 20:44, 17 December 2008 by >Zsakura2 (→‎Templates preparation)
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CES27k9bp library prep

Templates preparation

CES27K9bp-1 reaction system:
 E27kV2 (20nM):                         1ul         
 EconoTaq 2x master mix:               50ul       
 50X SYBG I                           0.4ul    
 100uM Ap1V4IU                        0.2ul     
 100uM AP2V4                          0.2ul
 H2O                                   50ul              
94C 2min -> (94C 45sec -> 60C 1min -> 72C 1min) x 18 -> 72C 3min -> 15C hold. Purified with Qiaquick columns. 
Result is 104nM dissovled in 50ul of H2O. Dilute it to 20nM with nonstick tube and 1X TE buffer. 

CES27K9bp-2 reaction system:                  x4
 CES27K 9bp (20nM):                     2ul         
 1.1X Platinum Taq SuperMix:           90ul       
 50X SYBG I                           0.6ul    
 100uM AP1V4IU                        0.4ul     
 100uM AP2V4                          0.4ul
 H2O                                    8ul              
94C 2min -> (94C 45sec -> 58C 1min -> 72C 1min) x 13 -> 72C 3min -> 15C hold. Purified with Qiaquick columns.
Result is 104nM dissovled in 60ul of H2O. Dilute it to 20nM with nonstick tube and 1X TE buffer.

PCR

  PCR system 1:                                  x48 
  Template (CES27K9bp-1):                0.1ul    9.6          
  EconoTaq 2x master mix:               50ul   2400
  50X SYBG I                           0.4ul   19.2    
  100uM AP1V41U                        0.1ul    4.8 
  100uM AP2V4                          0.1ul    4.8        
  H2O                                   50ul   2400   
  PCR system 2:                                  x48
  Template (CES27K9bp-1):                0.2ul     9.6          
  EconoTaw 2X master mix                  50ul    2400
  50X SYBG I                             0.4ul    19.2   
  100uM AP1V41U                          0.1ul     4.8    
  100uM AP2V4                            0.1ul     4.8        
  H2O                                     50ul    2400
  PCR system 2:                                  x48
  Template (CES27K9bp-2):                0.4ul    19.2          
  EconoTaw 2X master mix                  50ul    2400
  50X SYBG I                             0.4ul    19.2   
  100uM AP1V41U                          0.1ul     4.8    
  100uM AP2V4                            0.1ul     4.8        
  H2O                                     50ul    2400

94C 2min -> (94C 45sec -> 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).

Lambda exo digestion

  • To 150ul of DNA, add 14ul 10X lambda exo buffer, 2ul lambda exo.
  • 37C 1 hr -> 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

  • 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.