Kun:LabNotes/ASE/2008-1-3: Difference between revisions
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|- | |- | ||
| Template||8||1(20ng)||1(20ng)||1(50ng)||0.6|| | | 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 | | 10x buffer||1.5||1||1||1||1||1||1||1||1||1||1 | ||
Line 27: | Line 27: | ||
| Lib #5 (460nM)||1.8||||||||||1.8||||||||||1.8 | | Lib #5 (460nM)||1.8||||||||||1.8||||||||||1.8 | ||
|- | |- | ||
| H2O||0.9||6||6.5||5.4||5.75|| | | 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, | 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== | ==Exp.2 PCR on Hues6, GM20431, GC1F, GC1EP cDNA using the Hues6Ver2 primer set== | ||
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94C 3min -> (94C 30sec -> 56C 30sec -> 72C 30sec) x 40 -> 72C 3min -> 4C hold. | 94C 3min -> (94C 30sec -> 56C 30sec -> 72C 30sec) x 40 -> 72C 3min -> 4C hold. | ||
[[Image:2008-01-03-Hues6ver2-PCR.png|400px]] |
Latest revision as of 01:44, 5 January 2008
Exp.1 Capturing cSNPs with the eSNP84k probe set[edit]
Templates:
- Hues6 gDNA, 113ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
- Hues6 ds-cDNA, 155ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
- 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