Alice:LabNotes/2008-11-11
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CES27k9bp library prep[edit]
PCR[edit]
PCR system 1: x96 Template (CES27K9bp): 0.1ul 9.6 1.1X Platinum Taq supermix 90ul 7910 (7 tubes of 1130ul each) 50X SYBG I 0.4ul 39 100uM AP1V41U 0.2ul 19.2 100uM AP2V4 0.2ul 19.2 H2O 10ul 1703
PCR system 2: Template (CES27K9bp): 0.1ul 9.6 2X master mix 50ul 4800 (3 tubes + 1050ul) 50X SYBG I 0.4ul 39 100uM AP1V41U 0.2ul 19.2 100uM AP2V4 0.2ul 19.2 H2O 50ul 4800 94C 5min -> (95C 30sec -> 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). when wash buffer is used twice, the % yield is significantly lowered: 2-15.4 ng/ul *150ul 3-9.3ng/ul *150ul 4-13.5ng/ul*225ul 5-15.6ng/ul*150ul When it is only used for once, the results are different: 1-46.5ng/ul *225ul (260/280=1.87) 6-62.1ng/ul *150ul (260/280=1.9) 7-24.8ng/ul *150ul (260/280=1.7) 8-45.1ng/ul *150ul (260/280=1.9)
Lambda exo digestion[edit]
- To 150ul of DNA, add 20ul 10X lambda exo buffer, 10ul 10x lambda exo.
- 37C 2h. (alternatively: 37C 45min -> 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[edit]
- 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.
- Yield: 10ng/ul x 160ul
2D gel result: File:ZhangLab 2 2008-11-16 16hr 59min.jpg The gel on the right is the combination of all the product with low %yield after DNA concentrator step. File:ZhangLab 2 2008-11-16 22hr 14min.jpg File:ZhangLab 2 2008-11-16 22hr 46min.jpg