Kun:LabNotes/ASE/2007-11-11

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Exp.1 eSNPs capture (cont.)[edit]

Perform PCR on 5ul the capturing reaction on Hues6 gDNA, compare iTaq with JumpStart Taq.

                          x 1         x 2
     template:            5ul         10ul
     ABI 10x buffer:    4.5ul          9ul
     25mM MgCl2:          3ul          6ul
     10mM dNTP:           1ul          2ul
     100uM AmpF2Sol:   0.15ul        0.3ul
     100uM AmpRSol:    0.15ul        0.3ul
     50X SYBG I:        0.4ul        0.8ul
     iTaq:                1ul        2x1ul
     H2O:              35.3ul       70.6ul

94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 10 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.

PAGE purification of ds amplicons, 3 lanes per sample, two 5-well 6% TBE gel in total.

  • Hues6 gDNA: 40ul of iTaq amplified and 40ul of JumpStart amplified DNA (did today);
  • Hues6 cDNA: 16ul of each of the five subsets from the first batch of amplicons using iTaq;
  • Hues6 cDNA(no mouse): 16ul of each of the five subsets from the first batch of amplicons using iTaq;

PAGE quantification:

  File:2001-11-12-eSNP84k-Hues6-seqlib.png
  Hues6 gDNA:             14.5ng/ul = 468nM[WRONG] should be 181nM
  Hues6 cDNA:             11.5ng/ul = 371nM[WRONG] should be 144nM
  Hues6 no mouse cDNA:      15ng/ul = 484nM[WRONG] should be 187nM

Exp. 2 Repeat the capturing reactions with the eSNP84k probe set[edit]

Templates:

  1. Hues6 gDNA, 120ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
  2. Hues6 ds-cDNA, 155ng/ul, Lib #1: 2ng; Lib #2: 10ng; Lib #3 20ng; Lib #4: 100ng; Lib #5: 200ng
  3. Hues6(no mouse) ds-cDNA, 58ng/ul, Lib #1: 2ng; Lib #2: 10ng; Lib #3 20ng; Lib #4: 100ng; Lib #5: 200ng
gDNA Hues ds-cDNA Hues(no mouse) ds-cDNA
Template 6.7 1(2ng) 1(10ng) 1(20ng) 0.67 1.3 1(2ng) 1(10ng) 1(20ng) 1.8 3.6
10x buffer 2.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Lib #1 (384nM) 2 2 2
Lib #2 (580nM) 1.5 1.5 1.5
Lib #3 (190nM) 4.6 4.6 4.6
Lib #4 (500nM) 2.4 2.4 2.4
Lib #5 (460nM) 1.8 1.8 1.8
H2O 1 17 8.5 5.4 7.9 7.9 8 9.5 5.4 7.1 5.3

94C 3min->60C 12h-> add 2.5ul/1.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.