Alice:LabNotes/2008-12-15: Difference between revisions
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>Zsakura2 |
>Zsakura2 |
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*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: | *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=== |
Revision as of 18:19, 18 December 2008
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: 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 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
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
- 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.