AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions
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*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 |
Revision as of 00:34, 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,
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