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===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. [[image: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. [[image:2007-10-31-eSNP84k-probe-prep.jpg|350px]] 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. <b> 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.</b> [[Image:2007-11-02-eSNP84k-probe-quant.png|400px]] Estimated concentration: eSNP84k #1: 1ng/ul eSNP84k #2: 0.6ng/ul eSNP84k #3: 1.3ng/ul eSNP84k #4: 2ng/ul eSNP84k #5: 1.4ng/ul
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