AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions
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*Add 2 drops of mineral oil to prevent evaporation | *Add 2 drops of mineral oil to prevent evaporation | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style="background-color:#BFBFBF;font-size: | |- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" width="270" height="13" | ChIPK36me3 | |style="text-decoration:underline" width="270" height="13" | ChIPK36me3 | ||
| width="121" | Concentration ng/ul | | width="121" | Concentration ng/ul | ||
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| width="33" | Total | | width="33" | Total | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="13" | GM12878 | |style="text-decoration:none" height="13" | GM12878 | ||
| align="center" | 0.90 | | align="center" | 0.90 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | P10 | | height="13" | P10 | ||
| align="center" | 1.20 | | align="center" | 1.20 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | DF2 | | height="13" | DF2 | ||
| align="center" | 0.70 | | align="center" | 0.70 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H0 | | height="13" | H0 | ||
| align="center" | 1.00 | | align="center" | 1.00 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H7 | | height="13" | H7 | ||
| align="center" | 0.60 | | align="center" | 0.60 | ||
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| align="center" | 50 | | align="center" | 50 | ||
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| height="13" | H14 | | height="13" | H14 | ||
| align="center" | 0.50 | | align="center" | 0.50 | ||
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| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 0.60 | | align="center" | 0.60 | ||
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| align="center" | 50 | | align="center" | 50 | ||
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| height="13" | | | height="13" | | ||
| | | | ||
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| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" height="13" | ChIPK27me3 | |style="text-decoration:underline" height="13" | ChIPK27me3 | ||
| Concentration ng/ul | | Concentration ng/ul | ||
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| Total | | Total | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="13" | GM12878 | |style="text-decoration:none" height="13" | GM12878 | ||
| align="center" | 1.20 | | align="center" | 1.20 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | GM12878 Round 2 | | height="13" | GM12878 Round 2 | ||
| align="center" | 2.86 | | align="center" | 2.86 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="12" | P10 | | height="12" | P10 | ||
| align="center" | 1.30 | | align="center" | 1.30 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | DF2 | | height="13" | DF2 | ||
| align="center" | 2 | | align="center" | 2 | ||
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| align="center" | 50 | | align="center" | 50 | ||
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| height="13" | H0 | | height="13" | H0 | ||
| align="center" | 2.4 | | align="center" | 2.4 | ||
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| height="13" | H7 | | height="13" | H7 | ||
| align="center" | 1.2 | | align="center" | 1.2 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H14 | | height="13" | H14 | ||
| align="center" | 0.5 | | align="center" | 0.5 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 1.3 | | align="center" | 1.3 | ||
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| align="center" | 50 | | align="center" | 50 | ||
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| height="13" | | | height="13" | | ||
| | | | ||
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| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" height="13" | WCE | |style="text-decoration:underline" height="13" | WCE | ||
| Concentration ng/ul | | Concentration ng/ul | ||
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| Total | | Total | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="13" | GM12878 | |style="text-decoration:none" height="13" | GM12878 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | GM12878 Round 2 | | height="13" | GM12878 Round 2 | ||
| align="center" | 2.86 | | align="center" | 2.86 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | P10 | | height="13" | P10 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | P10 Round 2 | | height="13" | P10 Round 2 | ||
| align="center" | 2.86 | | align="center" | 2.86 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | DF2 | | height="13" | DF2 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | DF2 Round 2 | | height="13" | DF2 Round 2 | ||
| align="center" | 2.86 | | align="center" | 2.86 | ||
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| align="center" | 50 | | align="center" | 50 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H0 | | height="13" | H0 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H7 | | height="13" | H7 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H14 | | height="13" | H14 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 35.39 | | align="center" | 35.39 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | | | height="13" | | ||
| | | | ||
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| align="center" | | | align="center" | | ||
|- style="background-color:#BFBFBF;font-size: | |- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" height="13" | cDNA | |style="text-decoration:underline" height="13" | cDNA | ||
| Concentration ng/ul | | Concentration ng/ul | ||
Line 290: | Line 290: | ||
| Total | | Total | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="13" | GM | |style="text-decoration:none" height="13" | GM | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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| align="center" | 20 | | align="center" | 20 | ||
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| height="13" | H0 | | height="13" | H0 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H7 | | height="13" | H7 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H14 | | height="13" | H14 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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| align="center" | 20 | | align="center" | 20 | ||
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| | | | ||
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|- style="background-color:#BFBFBF;font-size: | |- style="background-color:#BFBFBF;font-size:8pt;font-weight:bold" align="center" | ||
|style="text-decoration:underline" height="13" | gDNA | |style="text-decoration:underline" height="13" | gDNA | ||
| Concentration ng/ul | | Concentration ng/ul | ||
Line 353: | Line 353: | ||
| Total | | Total | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
|style="text-decoration:none" height="13" | H7 | |style="text-decoration:none" height="13" | H7 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H14 | | height="13" | H14 | ||
| align="center" | 38.12 | | align="center" | 38.12 | ||
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| align="center" | 20 | | align="center" | 20 | ||
|- style="font-size: | |- style="font-size:8pt" align="center" | ||
| height="13" | H16 | | height="13" | H16 | ||
| align="center" | 38.12 | | align="center" | 38.12 |
Revision as of 00:40, 11 August 2012
Capturing Remaining Gimelbrant lab Sample
- Add 2 drops of mineral oil to prevent evaporation
ChIPK36me3 | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 0.90 | 40.00 | 0.45 | 2 | 7.6 | 50 |
P10 | 1.20 | 40.00 | 0.60 | 2 | 7.4 | 50 |
DF2 | 0.70 | 40.00 | 0.35 | 2 | 7.7 | 50 |
H0 | 1.00 | 40.00 | 0.50 | 2 | 7.5 | 50 |
H7 | 0.60 | 40.00 | 0.30 | 2 | 7.7 | 50 |
H14 | 0.50 | 40.00 | 0.25 | 2 | 7.8 | 50 |
H16 | 0.60 | 40.00 | 0.30 | 2 | 7.7 | 50 |
ChIPK27me3 | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 1.20 | 40.00 | 0.60 | 2 | 7.4 | 50 |
GM12878 Round 2 | 2.86 | 35.00 | 1.25 | 2 | 11.8 | 50 |
P10 | 1.30 | 40.00 | 0.65 | 2 | 7.4 | 50 |
DF2 | 2 | 40.00 | 1.00 | 2 | 7.0 | 50 |
H0 | 2.4 | 40.00 | 1.20 | 2 | 6.8 | 50 |
H7 | 1.2 | 40.00 | 0.60 | 2 | 7.4 | 50 |
H14 | 0.5 | 40.00 | 0.25 | 2 | 7.8 | 50 |
H16 | 1.3 | 40.00 | 0.65 | 2 | 7.4 | 50 |
WCE | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
GM12878 Round 2 | 2.86 | 35.00 | 1.25 | 2 | 11.8 | 50 |
P10 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
P10 Round 2 | 2.86 | 35.00 | 1.25 | 2 | 11.8 | 50 |
DF2 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
DF2 Round 2 | 2.86 | 35.00 | 1.25 | 2 | 11.8 | 50 |
H0 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H7 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H14 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H16 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
cDNA | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H0 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H7 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H14 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H16 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
gDNA | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
H7 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H14 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H16 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
- Make sure we have enough reagent before start,
- need 50ul probes, check
- need 66ul 10X Ampligase Buffer,
- need 33ul dNTP (10uM),
- need 13.2ul dNTP (10uM)
- need 13.2ul Stoffel (10U/ul)
- need 66ul Exo I (20U/ul)
- need 66ul ExoIII (200U/ul)
Prepare SLN Mix
- Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn | 1x | 16.5x |
dNTP (10uM) | 0.5 | 8.25 |
Stoffel (10U/ul) | 0.2 | 3.30 |
Ampligase (5U/ul) | 0.2 | 3.30 |
H2O | 0.1 | 1.65 |
SLN Mix for 50ul Rxn | 1x | 19.8x |
dNTP (10uM) | 1.25 | 24.75 |
Stoffel (10U/ul) | 0.5 | 9.90 |
Ampligase (5U/ul) | 0.5 | 9.90 |
H2O | 0.25 | 4.95 |
Prepare Exo I and III Mix
Exo I & III for 20ul Rxn | 1X | 16.5x |
Exo I (20U/ul) | 1 | 16.5 |
ExoIII (200U/ul) | 1 | 16.5 |
Exo I & III for 50ul Rxn | 1X | 19.8x |
Exo I (20U/ul) | 2.5 | 49.5 |
ExoIII (200U/ul) | 2.5 | 49.5 |
- When adding regents to the tube make sure you penetrate the oil layer
95C 2min -> -0.2C/sec to 60C -> 60C 24h -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III -> 37C 2h -> 90C 5min -> 4C hold