Alice:LabNotes/2008-12-15: Difference between revisions

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==CES27k9bp library prep==
==CES27k9bp library prep==
===Templates preparation===
===Templates preparation===
  CES27K9bp-1 reaction system:
 
  CES27K9bp-1 reaction system:                   x4
   E27kV2 (20nM):                        1ul         
   E27kV2 (20nM):                        1ul         
   EconoTaq 2x master mix:              50ul       
   EconoTaq 2x master mix:              50ul       
Line 19: Line 20:
   100uM AP2V4                          0.4ul
   100uM AP2V4                          0.4ul
   H2O                                    8ul               
   H2O                                    8ul               
 
  94C 2min -> (94C 45sec -> 58C 1min -> 72C 1min) x 13 -> 72C 3min -> 15C hold. Purified with Qiaquick columns.
  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.
  Result is 104nM dissovled in 60ul of H2O. Dilute it to 20nM with nonstick tube and 1X TE buffer.

Revision as of 20:44, 17 December 2008

CES27k9bp library prep

Templates preparation

CES27K9bp-1 reaction system:                    x4
 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.