Kun:LabNotes/ASE/2007-12-6
Exp.1 PCR on the pooled captured DNA
I still have some capturing reactions from 11-21-07, 11-26-07, 12-03-07 left. I decided to pool the reactions from the same templates togethers, and do PCR to make more Solexa sequencing library. The reason is that, based on the quantification experiment did yesterday, there are still not enough number of circularized molecules per target.
one reaction 8 reactions Template: 5~10ul each 10x Buffer: 9.0ul 72ul 25mM MgCl2 5.4ul 43.2ul 25mM dNTP 1.2ul 9.6ul 100uM AmpSolV6F2 0.4ul 3.2ul 100uM AmpSolV6R2 0.4ul 3.2ul iTaq 1ul 8ul 50X SYBG I 0.8ul 6.4ul H2O 72ul 576ul
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
Exp.2 Probe prep. for the CES22k set on two new libraries
Emily resynthesized two libraries for the CES22k set.
- CES22k 01783910053 (received Nov. 2007, same sequences as the above library) 100nM
- CES22k 10783810008 (received Nov. 2007, contains Solexa amplification adaptors) 100nM
PCR (with iTaq)
x1 x16x2 template(100nM) 0.1ul 2x0.4ul 10x ABI buffer 10ul 320ul 25mM MgCl2 6ul 192ul 10mM dNTP 2ul 64ul 100uM pAP1V6U 0.3ul 9.6ul 100uM phosAP2V6 0.3ul 9.6ul Platinium Taq 1ul 32ul H2O 80.8ul 2600ul
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Ethanol precipitation, resuspend in 135ul dH2O.
Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 100ul dH2O.
Dpn II & USER digestion: Add 13.5ul 10X Dpn II buffer, 5ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 8h, 75C 10min.