Revision as of 22:35, 18 August 2012 by >Alan6017518
Indexing PCR with Kapa SYBR Fast
- 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.
- Repeat PCR with same condition as first round of captured library in order to be consistance with the protocol
- 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
|
|
|
|
|
|
|
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 (sample 1-10) 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
Content (50ul Capture) 10 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
|
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
|
Purification & Quantification
Sample #
|
ID
|
Sample
|
1
|
ChIPK36me3
|
GM12878
|
2
|
ChIPK36me3
|
H0
|
3
|
ChIPK36me3
|
H7
|
4
|
ChIPK36me3
|
H14
|
5
|
ChIPK36me3
|
H16
|
6
|
ChIPK27me3
|
GM12878
|
7
|
ChIPK27me3
|
H0
|
8
|
ChIPK27me3
|
H7
|
9
|
ChIPK27me3
|
H14
|
10
|
ChIPK27me3
|
H16
|
11
|
WCE
|
GM12878
|
12
|
WCE
|
P10
|
13
|
WCE
|
DF2
|
14
|
WCE
|
H0
|
15
|
WCE
|
H7
|
16
|
WCE
|
H14
|
17
|
WCE
|
H16
|
18
|
cDNA
|
GM
|
19
|
cDNA
|
H0
|
20
|
cDNA
|
H7
|
21
|
cDNA
|
H14
|
22
|
cDNA
|
H16
|
23
|
gDNA
|
27
|
24
|
gDNA
|
28
|
25
|
gDNA
|
29
|
- Take 1ul to runa TBE PAGE gel
File:ZhangLab 2 2012-08-18 05hr 02min.jpg
File:ZhangLab 2 2012-08-18 05hr 00min.jpg
File:ZhangLab 2 2012-08-18 04hr 58min.jpg
- Qiaquick purify all libraries, elute with 30ul EB
- Qubit quantification
- The gel quantification is giving me negative values for a lot of the libraries
- I will just mix libraries based on their intensities at 180bp, and pool them based on the gel allocation, so we will have 3 set of libraries
Pool library Gel 1 (9 Libraries)
Sample
|
ng
|
Intensity
|
Volume ul
|
Total Intensity
|
100bp
|
1.25
|
12386
|
N/A
|
N/A
|
200bp
|
2.5
|
15800
|
N/A
|
N/A
|
400bp
|
5
|
19675
|
N/A
|
N/A
|
1
|
3.21
|
16508
|
3.75
|
61942
|
2
|
27.28
|
61942
|
1.00
|
61942
|
3
|
7.28
|
24180
|
2.56
|
61942
|
4
|
2.74
|
15627
|
3.96
|
61942
|
5
|
3.71
|
17455
|
3.55
|
61942
|
6
|
2.56
|
15279
|
4.05
|
61942
|
7
|
2.98
|
16079
|
3.85
|
61942
|
8
|
3.53
|
17109
|
3.62
|
61942
|
9
|
|
7480
|
8.28
|
61942
|
Pool library Gel 2
Sample
|
ng
|
Intensity
|
Volume ul
|
Total Intensity
|
100bp
|
1.25
|
6552
|
3.12
|
20465
|
200bp
|
2.50
|
9857
|
2.08
|
20465
|
400bp
|
5.00
|
11732
|
1.74
|
20465
|
10
|
6.32
|
13777
|
1.49
|
20465
|
11
|
|
3493
|
5.86
|
20465
|
12
|
|
3217
|
6.36
|
20465
|
13
|
2.27
|
8539
|
2.40
|
20465
|
14
|
|
4205
|
4.87
|
20465
|
15
|
|
5110
|
4.00
|
20465
|
16
|
|
3254
|
6.29
|
20465
|
17
|
11.50
|
20465
|
1.00
|
20465
|
Pool library Gel 3
Sample
|
ng
|
Intensity
|
Volume ul
|
Total Intensity
|
100bp
|
1.25
|
4274
|
1.43
|
6091
|
200bp
|
2.50
|
6091
|
1.00
|
6091
|
400bp
|
5.00
|
5827
|
1.05
|
6091
|
19
|
|
1738
|
3.51
|
6091
|
20
|
|
1619
|
3.76
|
6091
|
21
|
|
1665
|
3.66
|
6091
|
22
|
|
1994
|
3.05
|
6091
|
23
|
0.06
|
2545
|
2.39
|
6091
|
24
|
|
1517
|
4.02
|
6091
|
25
|
|
1616
|
3.77
|
6091
|