Kun:LabNotes/ASE/2008-1-3

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Exp.1 Capturing cSNPs with the eSNP84k probe set[edit]

Templates:

  1. Hues6 gDNA, 113ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
  2. Hues6 ds-cDNA, 155ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
  3. GC1F ds-cDNA, 48ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 200ng
Hues6g Hues6c GC1F
Template 8 1(20ng) 1(20ng) 1(50ng) 0.6 0.6 1(20ng) 1(20ng) 1(50ng) 2 4
10x buffer 1.5 1 1 1 1 1 1 1 1 1 1
Lib #1 (384nM) 2 2 2
Lib #2 (580nM) 1.5 1.5 1.5
Lib #3 (331nM) 2.6 2.6 2.6
Lib #4 (500nM) 2.4 2.4 2.4
Lib #5 (460nM) 1.8 1.8 1.8
H2O 0.9 6 6.5 5.4 5.75 6.6 6 6.5 5.4 5.72 3.2

94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 1mM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.

Exp.2 PCR on Hues6, GM20431, GC1F, GC1EP cDNA using the Hues6Ver2 primer set[edit]

Template:

  • Hues6 cDNA (04/18/07) 107ng/ul
  • GM20431 dsCDNA (12/31/07) 122ng/ul
  • GC1F sscDNA (06/05/07) 54ng/ul
  • GC1EP sscDNA(12/31/07) 114ng/ul

Make dilution to 5ng/ul

    Template:          1ul
    2X Taq Mastermix: 15ul
    2uM primers        3ul
    H2O:              11ul

94C 3min -> (94C 30sec -> 56C 30sec -> 72C 30sec) x 40 -> 72C 3min -> 4C hold.

 File:2008-01-03-Hues6ver2-PCR.png