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Alice:LabNotes/2008-12-15
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==CES27k9bp probe 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 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: [[Image:12-17-08 27k9bp.jpg]] reaction system 1 & 2: (note that system 2 never shoot up because of not enough template added) [[Image:12-16-08 27k9bp.jpg]] purified with ethanol precipitation, elute in 125ul H2O. ===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 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=== 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.
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