AlanFung:LabNotes/Capturing/2012-6-26
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- New sample list (some of the older concentration are just for calculations, the latest sample list ignored those)
- H7 and H14 were captured based on the wrong concentration, we used much less sample than expected
Capturing Reaction[edit]
- Add 2 drops of mineral oil to prevent evaporation
ChIPK36me3 | Sample Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
P10 | 40 | 0.6 | 5.00 | 4.40 | 50.00 |
DF2 | 40 | 0.6 | 5.00 | 4.40 | 50.00 |
ChIPK27me3 | Sample Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
P10 | 40.00 | 0.65 | 5.00 | 4.35 | 50.00 |
DF2 | 40.00 | 1.00 | 5.00 | 4.00 | 50.00 |
cDNA | Sample Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
P10E3 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
DF2 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
gDNA | Sample Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
P10E3 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
DF2 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
H0 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
GmgDNA:H0gDNA::1:1 | 5.25 | 2.49 | 2.00 | 10.26 | 20.00 |
Prepare SLN Mix[edit]
- Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn | 1x | 7.7x |
dNTP (10uM) | 0.5 | 3.85 |
Stoffel (10U/ul) | 0.2 | 1.54 |
Ampligase (5U/ul) | 0.2 | 1.54 |
H2O | 0.1 | 0.77 |
SLN Mix for 50ul Rxn | 1x | 4.4x |
dNTP (10uM) | 1.25 | 5.5 |
Stoffel (10U/ul) | 0.5 | 2.2 |
Ampligase (5U/ul) | 0.5 | 2.2 |
H2O | 0.25 | 1.1 |
Prepare Exo I and III Mix[edit]
Exo I & III for 20ul Rxn | 1X | 7.7x |
Exo I (20U/ul) | 1 | 7.7 |
ExoIII (200U/ul) | 1 | 7.7 |
Exo I & III for 50ul Rxn | 1X | 4.4x |
Exo I (20U/ul) | 2.5 | 11 |
ExoIII (200U/ul) | 2.5 | 11 |
- 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
Indexing qPCR[edit]
- Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn | Volume | Master Mix | ChIPK36me3 | IND |
Captured DNA | 27 | Do not add to MM | P10 Round 2 | 3 |
2X Kapa SYBG qPCR master mix | 50 | 220 | DF2 Round 2 | 4 |
10uM AmpF6.3Sol | 4 | 17.6 | ||
10uM AmpR6.3Ind | 4 | Do not add to MM | ChIPK27me3 | IND |
H2O | 15 | 66 | P10 Round 2 | 5 |
DF2 Round 2 | 6 | |||
Content (20ul Capture) 7rxn | Volume | Master Mix | ||
Captured DNA | 11 | Do not add to MM | cDNA | IND |
2X Kapa SYBG qPCR master mix | 50 | 385 | P10E3 | 7 |
10uM AmpF6.3Sol | 4 | 30.8 | DF2 | 8 |
10uM AmpR6.3Ind | 4 | Do not add to MM | ||
H2O | 31 | 238.7 | gDNA | IND |
GM | 9 | |||
P10E3 | 10 | |||
DF2 | 11 | |||
H0 | 12 | |||
GmgDNA:H0gDNA::1:1 | 13 |
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40 Monitor the reactions and terminate the program right before the amplification curves reach the plateau.
File:ZhangLab 2 2012-07-06 15hr 58min.jpg
- after talking to Kun, it appears that the larger amplicons are 2 circles forming when the polymerase for some reason didn't fall off and continue the amplification.
- Quantify all amplicons with qubit and pool to perform a size selection
- Qiaquick all samples elute with 30ul EB
gDNA | ng/ul | To get 180ng |
H7 | 7.24 | 13.81 |
H14 | 6.16 | 16.23 |
ChIPK36me3 | ng/ul | To get 180ng |
P10 | 14.42 | 6.93 |
DF2 | 16.08 | 6.22 |
ChIPK27me3 | ng/ul | To get 180ng |
P10 | 10.92 | 9.16 |
DF2 | 16.52 | 6.05 |
cDNA | ng/ul | To get 180ng |
P10E3 | 5.98 | 16.72 |
DF2 | 4.54 | 22.03 |
gDNA | ng/ul | To get 180ng |
GM | 9.72 | 10.29 |
P10E3 | 7.62 | 13.12 |
DF2 | 9.44 | 10.59 |
H0 | 8.28 | 12.08 |
GmgDNA:H0gDNA::1:1 | 12.5 | 8.00 |
- 15.47ng/ul after combine
Gel Size Selection[edit]
- 200ng/well is the limite for 5 well gel
- I will put 13ul of the pooled library ~200ng into each well