Revision as of 23:05, 25 August 2012 by >Noi
Bisulfite conversion
Lab IDs
|
Conc. (ng/ul)
|
Volume for 2000ng
|
LabID ID
|
H2O
|
Total amount (ng)
|
CT conversion reagent (ul)
|
Total volume (ul)
|
GL-1 |
295.8 |
6.76 |
GL-1 |
13.24 |
2000.00 |
130.00 |
150.00
|
GL-2 |
220.6 |
9.07 |
GL-2 |
10.93 |
2000.00 |
130.00 |
150.00
|
GL-3 |
149.7 |
13.36 |
GL-3 |
6.64 |
2000.00 |
130.00 |
150.00
|
GL-4 |
172.3 |
11.61 |
GL-4 |
8.39 |
2000.00 |
130.00 |
150.00
|
GL-5 |
285.2 |
7.01 |
GL-5 |
12.99 |
2000.00 |
130.00 |
150.00
|
GL-6 |
296.6 |
6.74 |
GL-6 |
13.26 |
2000.00 |
130.00 |
150.00
|
GL-7 |
320.0 |
6.25 |
GL-7 |
13.75 |
2000.00 |
130.00 |
150.00
|
GL-8 |
339.3 |
5.89 |
GL-8 |
14.11 |
2000.00 |
130.00 |
150.00
|
GL-9 |
467.5 |
4.28 |
GL-9 |
15.72 |
2000.00 |
130.00 |
150.00
|
GL-10 |
431.7 |
4.63 |
GL-10 |
15.37 |
2000.00 |
130.00 |
150.00
|
GL-11 |
349.1 |
5.73 |
GL-11 |
14.27 |
2000.00 |
130.00 |
150.00
|
GL-12 |
234.5 |
8.53 |
GL-12 |
11.47 |
2000.00 |
130.00 |
150.00
|
GL-13** |
72.2 |
20.00 |
GL-13 |
0.00 |
1444.80 |
130.00 |
150.00
|
GL-14 |
137.3 |
14.56 |
GL-14 |
5.44 |
2000.00 |
130.00 |
150.00
|
GL-15 |
207.5 |
9.64 |
GL-15 |
10.36 |
2000.00 |
130.00 |
150.00
|
GL-16 |
135.5 |
14.76 |
GL-16 |
5.24 |
2000.00 |
130.00 |
150.00
|
AL-1 |
226.6 |
8.83 |
AL-1 |
11.17 |
2000.00 |
130.00 |
150.00
|
AL-2 |
375.5 |
5.33 |
AL-2 |
14.67 |
2000.00 |
130.00 |
150.00
|
AL-3 |
256.8 |
7.79 |
AL-3 |
12.21 |
2000.00 |
130.00 |
150.00
|
AL-4 |
142.1 |
14.08 |
AL-4 |
5.92 |
2000.00 |
130.00 |
150.00
|
AL-5 |
140.1 |
14.27 |
AL-5 |
5.73 |
2000.00 |
130.00 |
150.00
|
330k BSPP capture set up
Subset ID
|
Adjusted Multiplier(2012_05_22)
|
Actual amount of probe required (probe:target ratio, 100:1)
|
Multiplier F
|
Probe concentration (ng/ul)
|
Volume (ul)
|
Volume for 23rxn (ul)
|
Subset ID
|
DMR.s1 |
0.25 |
2.24 |
0.56 |
26.2 |
0.02 |
0.49 |
DMR.s1
|
DMR.s2 |
0.35 |
2.24 |
0.78 |
30.1 |
0.03 |
0.60 |
DMR.s2
|
DMR.s3 |
1.15 |
18.90 |
21.74 |
21.15 |
1.03 |
23.64 |
DMR.s3
|
DMR.s4 |
1.00 |
18.90 |
18.90 |
22.34 |
0.85 |
19.46 |
DMR.s4
|
DMR.s5 |
4.83 |
18.90 |
91.29 |
29 |
3.15 |
72.40 |
DMR.s5
|
DMR.Exp1 |
1.00 |
18.90 |
18.90 |
31.88 |
0.59 |
13.64 |
DMR.Exp1
|
DMR.Exp2 |
1.00 |
18.90 |
18.90 |
24.11 |
0.78 |
18.03 |
DMR.Exp2
|
DMR.Exp3 |
1.00 |
18.90 |
18.90 |
28.9 |
0.65 |
15.04 |
DMR.Exp3
|
1.05uM Oligo suppressor-set1 |
|
|
|
|
0.125 |
2.88 |
1.05uM Oligo suppressor-set1
|
1.05uM Oligo suppressor-set2 |
|
|
|
|
0.125 |
2.88 |
1.05uM Oligo suppressor-set2
|
- This means that the maximum volume of bis-cvt gDNA is 42.15ul in total volume 55ul
Components
|
1rxn (ul)
|
23 rxn mix (ul)
|
10X AmpLigase Buffer |
5.50 |
126.50
|
DMR330k probe mix (ul) |
7.10 |
163.30
|
105nM Oligo suppressor |
0.25 |
5.75
|
Total volume (ul) |
12.85 |
295.55
|
Sample IDs
|
Conc. (ng/ul)
|
Volume for 250ng (ul)
|
Sample IDs
|
H2O
|
Total amount of bis-cvt gDNA (ng)
|
Probe mix (ul)
|
10X AmpLigase Buffer
|
1.05uM Oligo suppressor set1 &2 (ul)
|
Total volume (ul)
|
GL-1 |
6.30 |
39.68 |
GL-1 |
2.47 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-2 |
5.00 |
42.15 |
GL-2 |
0.00 |
210.75 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-3 |
8.92 |
28.03 |
GL-3 |
14.12 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-4 |
7.14 |
35.01 |
GL-4 |
7.14 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-5* |
5.34 |
42.15 |
GL-5* |
0.00 |
225.08 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-6## |
3.97 |
42.15 |
GL-6## |
0.00 |
167.34 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-7* |
5.30 |
42.15 |
GL-7* |
0.00 |
223.40 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-8* |
5.08 |
42.15 |
GL-8* |
0.00 |
214.12 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-9 |
5.51 |
42.15 |
GL-9 |
0.00 |
232.25 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-10 |
5.31 |
42.15 |
GL-10 |
0.00 |
223.82 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-11 |
4.99 |
42.15 |
GL-11 |
0.00 |
210.33 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-12 |
4.97 |
42.15 |
GL-12 |
0.00 |
209.49 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-13 |
7.11 |
35.16 |
GL-13 |
6.99 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-14 |
7.27 |
34.39 |
GL-14 |
7.76 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-15 |
8.01 |
31.21 |
GL-15 |
10.94 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
GL-16 |
7.25 |
34.48 |
GL-16 |
7.67 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
AL-1 |
5.06 |
42.15 |
AL-1 |
0.00 |
213.28 |
7.10 |
5.50 |
0.25 |
55.00
|
AL-2 |
5.00 |
42.15 |
AL-2 |
0.00 |
210.75 |
7.10 |
5.50 |
0.25 |
55.00
|
AL-3 |
5.22 |
42.15 |
AL-3 |
0.00 |
220.02 |
7.10 |
5.50 |
0.25 |
55.00
|
AL-4 |
7.16 |
34.92 |
AL-4 |
7.23 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
AL-5 |
8.40 |
29.76 |
AL-5 |
12.39 |
250.00 |
7.10 |
5.50 |
0.25 |
55.00
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (08.24.2012, ~5pm)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN mix solution
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume72ul
|
AmpliTaq Stoffel |
10 |
U/ul |
2 |
U/ul |
14.40
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
7.20
|
dNTP |
1000 |
uM |
100 |
uM |
7.20
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
7.20
|
H2O |
|
|
|
|
36.00
|
Total |
|
|
|
|
72.00
|
- Add 3ul of SLN mix to each