Kun:LabNotes/ASE/2007-10-29
Exp1: Probe prep (cont.)
- Starting from ethanol precipitated DNAs made at 10/18/2007.
- Pool two tubes of the same amplicons into ~100ul in total, add 11ul 10x Lambda Exo buffer, 4ul 5U/ul Lambda exonuclease. Incubate @ 37C for 2 hours, check 2ul of the products in both 8% TBE gels and 6% TBU gels.
8% TBE gel 6% TBU gel File:2007-10-29-PCR-Lambda exo-TBE.jpg File:2007-10-29-PCR-Lambda exo-TBU.jpg
- I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours. I probably used the wrong reverse primer for the eSNP84k sets. I should have used the phosAP2V4 instead of AP2V4, since lambda exonuclease doesn't cut without a 5'-phosphate group.
Exp2: Repeat PCR amplification on the eSNP84k library
Reactions eSNP84k set1-5 (100nM):
x 8 x 5 Template 0.2ul 1.6ul x 5 10X buffer 10ul 80ul x 5 10mM dNTP 2ul 16ul x 5 100uM AP1V41U 0.4ul 3.2ul x 5 100uM phosAp2V4 0.4ul 3.2ul x 5 AmpliTaq Gold 1ul 16ul x 5 H2O 86ul 680ul x 5 50X SYBR I 0.8ul 6.4ul x 5
95C 15min -> 17 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold
The curves came up a little bit later than the previous amplification using JumpStart Taq. I did 3 more cycles, and purified 200ul of reactions with QiaQuick columns. The yield was pretty low, so I added two more cycles and purified another 200ul reactions.
Lib 20cyc 22cyc #1 10ng/ul 19ng/ul #2 8ng/ul 15ng/ul #3 7ng/ul 15ng/ul #4 10ng/ul 24ng/ul #5 6ng/ul 10ng/ul
I also purified the remaining reactions (400ul each) with ethanol precipitation.
Mix the DNAs of the same library, add 10ul NEBuffer 4, 2ul BciVI, incubate @ 37C for 2 hours, 65C 20min.
File:2007-10-30-padlock intermediate.jpg
Add 2ul T7 exonuclease, 37C 2h, add 2ul USER enzyme, 37C 2h.
For the ethanol precipitated DNAs, I resuspended them in 90ul dH2O, add 10ul 10x NEBuffer 4, 2ul BciVI, 37C 2h, 65C 20min, add 2ul T7 exo, 37C 2h.
File:2007-10-31-eSNP84k-probe-prep.jpg
For the batch I, add 3ul USER, 37C 2h. For the batch II, incubate @ 37C 2h, add 5ul USER, incubate @ 37C 4h. Check with PAGE gel.
Perform phenol extraction on the batch I, then concentrate with Vacufuge. I lost all DNA in the vacufuge because I placed the rotor upside down and all liquid came out during spinning. I've turned the rotor over.