Revision as of 00:38, 11 July 2012 by >Alan6017518
- 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
Always up to date sample list
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
|
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