Revision as of 04:12, 12 August 2012 by >Alan6017518
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,
- need 33ul dNTP (10uM),
- need 13.2ul dNTP (10uM)
- need 13.2ul Stoffel (10U/ul)
- need 66ul Exo I (20U/ul)
- need 66ul ExoIII (200U/ul)
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
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