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

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==CES27k9bp library prep==
==CES27k9bp probe prep==
===Templates preparation===
===Templates preparation===


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  reaction system 1 & 2: (note that system 2 never shoot up because of not enough template added)
  reaction system 1 & 2: (note that system 2 never shoot up because of not enough template added)
  [[Image:12-16-08 27k9bp.jpg]]
  [[Image:12-16-08 27k9bp.jpg]]
purified with ethanol precipitation, elute in 125ul H2O.


Purified one plate with three DNA concentrator-100 columns, eluted with 150ul EB (only use wash buffer once).
===Lambda exo digestion===
reaction system 1:
*To 125ul of DNA, add 14ul 10X lambda exo buffer, 2ul lambda exo.
*37C 1 hr -> 75C 15 min
*Purified the digestion Qiaquick column, eluted with 50ul ddH2O
*Yield: 21.9ng/ul x 50ul 260/280=1.91


===Lambda exo digestion===
 
*To 150ul of DNA, add 14ul 10X lambda exo buffer, 2ul lambda exo.  
reaction system 3&4:
*To 125ul of DNA, add 14ul 10X lambda exo buffer, 3ul lambda exo.  
*37C 1 hr -> 75C 15 min
*37C 1 hr -> 75C 15 min
*Purified the digestion Qiaquick columns, eluted with 80ul ddH2O, combine the elution.
*Purified the digestion Qiaquick columns, eluted with 80ul ddH2O, combine the elution.
*Yield: 105ng/ul x 750ul (all products combined)
Yield: 81.6ng/ul * 80ul           
      72.8ng/ul * 80ul                 
      260/280=1.90 for both


===Dpn II & USER enzyme digestion===
===Dpn II & USER enzyme digestion===
*To 80ul of ssDNA, add 5ul USER enzyme, 37C 8hr.
reaction system 1:
*Add 10ul 10X DpnII buffer, 5ul 100uM DpnII-V4 guide oligo
*To 50ul of ssDNA, add 1.5ul USER enzyme, 37C 1hr.
*94C5min -> 60C 5min -> turn off and wait for 20min -> add 5ul Dpn II -> 37C 2h.
*Add 6ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
*Add 5ul USER enzyme -> 37C 2h -> 75C 20min
*94C5min -> 60C 5min -> turn off and wait for 20min -> add 1ul Dpn II (50K/ml)-> 37C 1h.
*(alternatively, all the above methods can be done for 37C 1 hr, the 2D gel result looked same for both methods)
*Add 1ul USER enzyme -> 37C 1h -> 75C 20min
*PAGE purification in 6% TB-Urea 2D gels.
*PAGE purification in 6% TB-Urea 2D gels.
*DNA were eluted from the shearing gel assembly and Nanosep columns.
*DNA were eluted from the shearing gel assembly and Nanosep columns.
*Ethanol purification and PAGE gel quantification.
*Ethanol purification and PAGE gel quantification.
reaction system 3&4:
*To 80ul of ssDNA, add 4ul USER enzyme, 37C 1hr.
*Add 10ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
*94C5min -> 60C 5min -> turn off and wait for 20min -> add 2ul Dpn II (50K/ml) -> 37C 1h.
*Add 2ul USER enzyme -> 37C 1h -> 75C 20min
*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: 5ng/ul *80ul
I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working.
Therefore half of the plate is actually not working, thus final percent yield is too low.

Latest revision as of 21:03, 21 December 2008

CES27k9bp probe prep[edit]

Templates preparation[edit]

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[edit]

  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 has the same protocol except the use of CES27K9bp-2 template    
  PCR system 3:                                  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 4:                                  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.
PCR result:
reaction system 3 & 4:
File:12-17-08 27k9bp.jpg

reaction system 1 & 2: (note that system 2 never shoot up because of not enough template added)
File:12-16-08 27k9bp.jpg

purified with ethanol precipitation, elute in 125ul H2O.

Lambda exo digestion[edit]

reaction system 1:

  • To 125ul of DNA, add 14ul 10X lambda exo buffer, 2ul lambda exo.
  • 37C 1 hr -> 75C 15 min
  • Purified the digestion Qiaquick column, eluted with 50ul ddH2O
  • Yield: 21.9ng/ul x 50ul 260/280=1.91


reaction system 3&4:

  • To 125ul of DNA, add 14ul 10X lambda exo buffer, 3ul lambda exo.
  • 37C 1 hr -> 75C 15 min
  • Purified the digestion Qiaquick columns, eluted with 80ul ddH2O, combine the elution.
Yield: 81.6ng/ul * 80ul            
      72.8ng/ul * 80ul                   
      260/280=1.90 for both

Dpn II & USER enzyme digestion[edit]

reaction system 1:

  • To 50ul of ssDNA, add 1.5ul USER enzyme, 37C 1hr.
  • Add 6ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
  • 94C5min -> 60C 5min -> turn off and wait for 20min -> add 1ul Dpn II (50K/ml)-> 37C 1h.
  • Add 1ul USER enzyme -> 37C 1h -> 75C 20min
  • 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.

reaction system 3&4:

  • To 80ul of ssDNA, add 4ul USER enzyme, 37C 1hr.
  • Add 10ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
  • 94C5min -> 60C 5min -> turn off and wait for 20min -> add 2ul Dpn II (50K/ml) -> 37C 1h.
  • Add 2ul USER enzyme -> 37C 1h -> 75C 20min
  • 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: 5ng/ul *80ul
I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working. 
Therefore half of the plate is actually not working, thus final percent yield is too low.