Revision as of 09:28, 18 August 2012 by >Alan6017518
Capturing Remaining Gimelbrant lab Sample
- Add 2 drops of mineral oil to prevent evaporation
- Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
ChIPK36me3
|
Concentration ng/ul
|
Vol
|
Vol of probes
|
10X Ampligase Buffer
|
H2O
|
Total
|
GM12878
|
0.90
|
40.00
|
0.45
|
2
|
7.6
|
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
|
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
|
P10
|
35.39
|
5.65
|
2.49
|
2
|
9.9
|
20
|
DF2
|
35.39
|
5.65
|
2.49
|
2
|
9.9
|
20
|
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
|
- When adding regents to the tube make sure you penetrate the oil layer
Store under βCES36K
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
Prepare SLN Mix
- Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn
|
1x
|
16x
|
dNTP (10uM)
|
0.5
|
8
|
Stoffel (10U/ul)
|
0.2
|
3.2
|
Ampligase (5U/ul)
|
0.2
|
3.2
|
H2O
|
0.1
|
1.6
|
|
|
|
SLN Mix for 50ul Rxn
|
1x
|
11x
|
dNTP (10uM)
|
1.25
|
13.75
|
Stoffel (10U/ul)
|
0.5
|
5.5
|
Ampligase (5U/ul)
|
0.5
|
5.5
|
H2O
|
0.25
|
2.75
|
Prepare Exo I and Exo III Mix
Exo I & III for 20ul Rxn
|
1X
|
16x
|
Exo I (20U/ul)
|
1
|
16
|
ExoIII (200U/ul)
|
1
|
16
|
|
|
|
Exo I & III for 50ul Rxn
|
1X
|
11x
|
Exo I (20U/ul)
|
2.5
|
6.25
|
ExoIII (200U/ul)
|
2.5
|
6.25
|
Indexing qPCR
- Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn
|
Volume
|
Master Mix
|
ChIPK36me3
|
Index
|
ChIPK27me3
|
Index
|
|
|
Captured DNA
|
27
|
Do not add to MM
|
GM12878
|
1
|
GM12878
|
10
|
|
|
2X Kapa HiFi mix
|
50
|
550
|
H0
|
2
|
H0
|
11
|
|
|
10uM AmpF6.3Sol
|
4
|
44
|
H7
|
3
|
H7
|
12
|
|
|
10uM AmpR6.3Ind
|
4
|
Do not add to MM
|
H14
|
4
|
H14
|
13
|
|
|
H2O
|
15
|
165
|
H16
|
9
|
H16
|
14
|
|
|
SYBR Green 50X
|
0.4
|
4.4
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Content (20ul Capture) 7rxn
|
Volume
|
Master Mix
|
WCE
|
Index
|
cDNA
|
Index
|
gDNA
|
Index
|
Captured DNA
|
11
|
Do not add to MM
|
GM12878
|
15
|
GM
|
22
|
H7
|
27
|
2X Kapa SYBG qPCR master mix
|
50
|
825
|
P10
|
16
|
H0
|
23
|
H14
|
28
|
10uM AmpF6.3Sol
|
4
|
66
|
DF2
|
17
|
H7
|
24
|
H16
|
29
|
10uM AmpR6.3Ind
|
4
|
Do not add to MM
|
H0
|
18
|
H14
|
25
|
|
|
H2O
|
31
|
511.5
|
H7
|
19
|
H16
|
26
|
|
|
SYBR Green 50X
|
0.4
|
6.6
|
H14
|
20
|
|
|
|
|
|
|
|
H16
|
21
|
|
|
|
|
95C 2min -> (98C 20S -> 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.
Indexing PCR for 3 samples
Indexing PCR with Kapa SYBR Fast
- Repeat PCR with same condition as first round of captured library in order to be consistance
- Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
- Elute with 30ul EB, use 15ul for amplification
Content (50ul Capture) 9 rxn
|
Volume
|
Master Mix
|
ChIPK36me3
|
Index
|
ChIPK27me3
|
Index
|
|
|
Captured DNA
|
27
|
Do not add to MM
|
GM12878
|
1
|
GM12878
|
10
|
|
|
2X Kapa SYBG qPCR master mix
|
50
|
495
|
H0
|
2
|
H0
|
11
|
|
|
10uM AmpF6.3Sol
|
4
|
39.6
|
H7
|
3
|
H7
|
12
|
|
|
10uM AmpR6.3Ind
|
4
|
Do not add to MM
|
H14
|
4
|
H14
|
13
|
|
|
H2O
|
15
|
148.5
|
H16
|
9
|
H16
|
14
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Content (20ul Capture) 13rxn
|
Volume
|
Master Mix
|
WCE
|
Index
|
cDNA
|
Index
|
gDNA
|
Index
|
Captured DNA
|
11
|
Do not add to MM
|
GM12878
|
15
|
GM
|
22
|
H7
|
27
|
2X Kapa SYBG qPCR master mix
|
50
|
715
|
P10
|
16
|
H0
|
23
|
H14
|
28
|
10uM AmpF6.3Sol
|
4
|
57.2
|
DF2
|
17
|
H7
|
24
|
H16
|
29
|
10uM AmpR6.3Ind
|
4
|
Do not add to MM
|
H0
|
18
|
H14
|
25
|
|
|
H2O
|
31
|
443.3
|
H7
|
19
|
H16
|
26
|
|
|
|
|
|
H14
|
20
|
|
|
|
|
|
|
|
H16
|
21
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Content (15ul Capture) 3rxn
|
Volume
|
Master Mix
|
Type
|
Sample
|
Index
|
|
|
|
Captured DNA
|
15
|
Do not add to MM
|
ChIPK36me3
|
H0
|
2
|
|
|
|
2X Kapa SYBG qPCR master mix
|
50
|
165
|
WCE
|
DF2
|
17
|
|
|
|
10uM AmpF6.3Sol
|
4
|
13.2
|
cDNA
|
GM
|
22
|
|
|
|
10uM AmpR6.3Ind
|
4
|
Do not add to MM
|
|
|
|
|
|
|
H2O
|
27
|
89.1
|
|
|
|
|
|
|
- The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
- First add Index primer
- Then add captured DNA
- Prepare Master Mix
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.
- Master mix for 50ul reaction was frozen on the ice block, I realized this after adding in the MM to wells, ran pcr nothing amplified based on the PCR curve.
- Qiaquick elute with 29ul
- Redo amplification
Purification & Quantification
- Qiaquick purify all libraries, elute with 30ul EB
- Qubit quantification