Kun:LabNotes/ASE/2007-10-29

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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.