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.