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.

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