Kun:LabNotes/ASE/2007-11-20: Difference between revisions
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(3 intermediate revisions by the same user not shown) | |||
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{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
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| align="center" style="background:#f0f0f0;"|''' ''' | |||
| align="center" style="background:#f0f0f0;"|''' ''' | | align="center" style="background:#f0f0f0;"|''' ''' | ||
| align="center" style="background:#f0f0f0;"|'''Template''' | | align="center" style="background:#f0f0f0;"|'''Template''' | ||
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| align="center" style="background:#f0f0f0;"|'''H2O''' | | align="center" style="background:#f0f0f0;"|'''H2O''' | ||
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| GM20431 gDNA(347ng/ul)||2.3ul||1ul||5.4ul||0.3ul | |1||GM20431 gDNA(347ng/ul)||2.3ul||1ul||5.4ul||0.3ul | ||
|- | |- | ||
| GM20431 cDNA (121ng/ul)||0.4ul||1ul||5.4ul||2.2ul | |2||GM20431 cDNA (121ng/ul)||0.4ul||1ul||5.4ul||2.2ul | ||
|- | |- | ||
| GC1F cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul | |3||GC1F cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul | ||
|- | |- | ||
| GC1EP cDNA(52ng/ul)||1ul||1ul||5.4ul||1.6ul | |4||GC1EP cDNA(52ng/ul)||1ul||1ul||5.4ul||1.6ul | ||
|- | |- | ||
| Hues6 gDNA(113ng/ul)||2.6ul||1ul||5.4ul||0ul | |5||Hues6 gDNA(113ng/ul)||2.6ul||1ul||5.4ul||0ul | ||
|- | |- | ||
| Hues6 cDNA(155ng/ul)||0.3ul||1ul||5.4ul||2.3ul | |6||Hues6 cDNA(155ng/ul)||0.3ul||1ul||5.4ul||2.3ul | ||
|- | |- | ||
| H1 gDNA(222ng/ul)||2.6ul||1ul||5.4ul||0ul | |7||H1 gDNA(222ng/ul)||2.6ul||1ul||5.4ul||0ul | ||
|- | |- | ||
| H1 cDNA(35ng/ul)||1.4ul||1ul||5.4ul||1.2ul | |8||H1 cDNA(35ng/ul)||1.4ul||1ul||5.4ul||1.2ul | ||
|- | |- | ||
| H2 gDNA(200ng/ul)||2.6ul||1ul||5.4ul||0ul | |9||H2 gDNA(200ng/ul)||2.6ul||1ul||5.4ul||0ul | ||
|- | |- | ||
| H2 cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul | |10||H2 cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul | ||
|- | |- | ||
| BJ MDA DNA(576ng/ul) ||2.6ul||1ul||5.4ul||0ul | |11||BJ MDA DNA(576ng/ul) ||2.6ul||1ul||5.4ul||0ul | ||
|- | |- | ||
| BJ cDNA(18ng/ul)||2.6ul||1ul||5.4ul||0ul | |12||BJ cDNA(18ng/ul)||2.6ul||1ul||5.4ul||0ul | ||
|} | |} | ||
94C 5min->60C 18h-> add 0.5ul 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 | 94C 5min->60C 18h-> add 0.5ul 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 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold. | ||
[11-22-2007] PCR | |||
one reaction 12 reactions | |||
Template: 5ul each | |||
10x Buffer: 9.5ul 123.5ul | |||
25mM MgCl2 6ul 78ul | |||
25mM dNTP 1.2ul 15.6ul | |||
100uM AmpSolV6F2 0.4ul 5.2ul | |||
100uM AmpSolV6R2 0.4ul 5.2ul | |||
Platinium Taq 1ul 13ul | |||
50X SYBG I 0.8ul 10.4ul | |||
H2O 76.7ul 997ul | |||
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. | |||
Added 3 more cycles for #3, 4, 6, 8, 10, 11, 12. | |||
[[Image:2007-11-24-CES22k-capture-PCR.png|500px]] | |||
Since the amplification curve for #1 came up much earlier than others, I repeat the PCR on 3ul #1 with 10 cycles at the second stage of amplification. |
Latest revision as of 23:12, 24 November 2007
Exp. 1 Perform MDA on the gDNA from BJ Fibroblast[edit]
Template DNA(6ng/ul) 20ul 10X RepliPhi buffer 2.5ul 1mM N6 2.5ul
95C 3min -> chill on ice -> add the reaction mix: 10X RepliPhi buffer 8.5ul 25mM dNTP 4ul 1mM N6 2.5ul H2O 49ul 100mM DTT 5ul RepliPhi phi-29 5ul 30C 3h -> 85C 10min Purify with Microcon YM-100 => 576ng/ul
Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples[edit]
Template | 10x buffer | CES22k probes (277nM) | H2O | ||
1 | GM20431 gDNA(347ng/ul) | 2.3ul | 1ul | 5.4ul | 0.3ul |
2 | GM20431 cDNA (121ng/ul) | 0.4ul | 1ul | 5.4ul | 2.2ul |
3 | GC1F cDNA(48ng/ul) | 1ul | 1ul | 5.4ul | 1.6ul |
4 | GC1EP cDNA(52ng/ul) | 1ul | 1ul | 5.4ul | 1.6ul |
5 | Hues6 gDNA(113ng/ul) | 2.6ul | 1ul | 5.4ul | 0ul |
6 | Hues6 cDNA(155ng/ul) | 0.3ul | 1ul | 5.4ul | 2.3ul |
7 | H1 gDNA(222ng/ul) | 2.6ul | 1ul | 5.4ul | 0ul |
8 | H1 cDNA(35ng/ul) | 1.4ul | 1ul | 5.4ul | 1.2ul |
9 | H2 gDNA(200ng/ul) | 2.6ul | 1ul | 5.4ul | 0ul |
10 | H2 cDNA(48ng/ul) | 1ul | 1ul | 5.4ul | 1.6ul |
11 | BJ MDA DNA(576ng/ul) | 2.6ul | 1ul | 5.4ul | 0ul |
12 | BJ cDNA(18ng/ul) | 2.6ul | 1ul | 5.4ul | 0ul |
94C 5min->60C 18h-> add 0.5ul 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 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.
[11-22-2007] PCR
one reaction 12 reactions Template: 5ul each 10x Buffer: 9.5ul 123.5ul 25mM MgCl2 6ul 78ul 25mM dNTP 1.2ul 15.6ul 100uM AmpSolV6F2 0.4ul 5.2ul 100uM AmpSolV6R2 0.4ul 5.2ul Platinium Taq 1ul 13ul 50X SYBG I 0.8ul 10.4ul H2O 76.7ul 997ul
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
Added 3 more cycles for #3, 4, 6, 8, 10, 11, 12. File:2007-11-24-CES22k-capture-PCR.png Since the amplification curve for #1 came up much earlier than others, I repeat the PCR on 3ul #1 with 10 cycles at the second stage of amplification.