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:
- Hues6 gDNA, 120ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
- Hues6 ds-cDNA, 155ng/ul, Lib #1: 2ng; Lib #2: 10ng; Lib #3 20ng; Lib #4: 100ng; Lib #5: 200ng
- 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.