Revision as of 00:48, 5 June 2014 by >Noi
MONOD V1N3 BSPP capture (repeat sensitivity test)
Link to calendar: [[1]]
- I repeated MONOD V1N3 capture experiment on 3014-05-29: [[2]]
- I reduced capture volume to 10ul.
- I reduced incubation time after adding SLN mix between 4-6h since I think that 20h is not necessary for polymerization and may cause more back ground if DNA template level is too low.
- I will add experimental procedures later
Bisulfite conversion
Mix
|
82.1ng/ul JK DNA
|
H2O
|
Aliquot
|
25ng |
1.28 |
82.72 |
20.00
|
50ng |
2.56 |
81.44 |
20.00
|
100ng |
5.12 |
78.88 |
20.00
|
200ng |
10.23 |
73.77 |
20.00
|
Sample
|
Conc. (ng/ul)
|
Amount (ng)
|
Volume (ul)
|
H2O (ul)
|
Lightnig Conversion reagent
|
Total
|
JK-1 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-2 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-3 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-4 |
82.1 |
25 |
0.30 |
19.70 |
130 |
150.00
|
JK-5 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-6 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-7 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-8 |
82.1 |
50 |
0.61 |
19.39 |
130 |
150.00
|
JK-9 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-10 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-11 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-12 |
82.1 |
100 |
1.22 |
18.78 |
130 |
150.00
|
JK-13 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-14 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-15 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
JK-16 |
82.1 |
200 |
2.44 |
17.56 |
130 |
150.00
|
- Elute with 10ul Elution buffer
- Use 1ul for Qubit ssDNA assay
Qubit ssDNA assay results
- I did many bisulfite conversion reactions to compare the recovery rate with different inputs
Sample
|
Conc. in the Qubit
|
Unit
|
uL used
|
Dilution
|
Sample conc. (ng/ul)
|
Yield in 9ul (ng)
|
Input DNA (ng)
|
% Recovery
|
JK-1 |
3.8 |
ng/mL |
1 |
200 |
0.760 |
6.84 |
25 |
27.36
|
JK-2 |
4.9 |
ng/mL |
1 |
200 |
0.980 |
8.82 |
25 |
35.28
|
JK-3 |
2.5 |
ng/mL |
1 |
200 |
0.500 |
4.50 |
25 |
18.00
|
JK-4 |
5.11 |
ng/mL |
1 |
200 |
1.022 |
9.20 |
25 |
36.79
|
JK-5 |
10.8 |
ng/mL |
1 |
200 |
2.160 |
19.44 |
50 |
38.88
|
JK-6 |
9.06 |
ng/mL |
1 |
200 |
1.812 |
16.31 |
50 |
32.62
|
JK-7 |
12.3 |
ng/mL |
1 |
200 |
2.460 |
22.14 |
50 |
44.28
|
JK-8 |
11.2 |
ng/mL |
1 |
200 |
2.240 |
20.16 |
50 |
40.32
|
JK-9 |
25 |
ng/mL |
1 |
200 |
5.000 |
45.00 |
100 |
45.00
|
JK-10 |
26.7 |
ng/mL |
1 |
200 |
5.340 |
48.06 |
100 |
48.06
|
JK-11 |
28.4 |
ng/mL |
1 |
200 |
5.680 |
51.12 |
100 |
51.12
|
JK-12 |
24.3 |
ng/mL |
1 |
200 |
4.860 |
43.74 |
100 |
43.74
|
JK-13 |
90.1 |
ng/mL |
1 |
200 |
18.020 |
162.18 |
200 |
81.09
|
JK-14 |
89 |
ng/mL |
1 |
200 |
17.800 |
160.20 |
200 |
80.10
|
JK-15 |
79.7 |
ng/mL |
1 |
200 |
15.940 |
143.46 |
200 |
71.73
|
JK-16 |
85.4 |
ng/mL |
1 |
200 |
17.080 |
153.72 |
200 |
76.86
|
BSPP capture set up
strip 1
|
5:500
|
5:500
|
10:500
|
10:500
|
25:500
|
25:500
|
50:500
|
50:500
|
BIS-DNA required (ng) |
5 |
5 |
10 |
10 |
25 |
25 |
50 |
50
|
JK # |
JK-1 |
JK-2 |
JK-5 |
JK-6 |
JK-9 |
JK-10 |
JK-13 |
JK-14
|
Conc. |
0.760 |
0.980 |
2.160 |
1.812 |
5.000 |
5.340 |
18.020 |
17.800
|
Volume (ul) |
6.58 |
5.10 |
4.63 |
5.52 |
5.00 |
4.68 |
2.77 |
2.81
|
H2O (ul) |
0.27 |
1.75 |
2.07 |
1.18 |
1.25 |
1.57 |
2.72 |
2.69
|
V1N3 amount (ng) |
0.11 |
0.11 |
0.22 |
0.22 |
0.55 |
0.55 |
1.10 |
1.10
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.15 |
0.15 |
0.30 |
0.30 |
0.75 |
0.75 |
1.50 |
1.50
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
strip 2
|
5:1000
|
5:1000
|
10:1000
|
10:1000
|
25:1000
|
25:1000
|
50:1000
|
50:1000
|
BIS-DNA required (ng) |
5 |
5 |
10 |
10 |
25 |
25 |
50 |
50
|
JK # |
JK-4 |
JK-5 |
JK-7 |
JK-8 |
JK-11 |
JK-12 |
JK-15 |
JK-16
|
Conc. |
1.022 |
2.160 |
2.460 |
2.240 |
5.680 |
4.860 |
15.940 |
17.080
|
Volume (ul) |
4.89 |
2.31 |
4.07 |
4.46 |
4.40 |
5.14 |
3.14 |
2.93
|
H2O (ul) |
1.81 |
4.38 |
2.33 |
1.93 |
1.09 |
0.35 |
0.85 |
1.06
|
V1N3 amount (ng) |
0.22 |
0.22 |
0.44 |
0.44 |
1.10 |
1.10 |
2.20 |
2.20
|
V1N3 conc. (ng/ul) |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73 |
0.73
|
V1N3 volume (ul) |
0.30 |
0.30 |
0.60 |
0.60 |
1.50 |
1.50 |
3.01 |
3.01
|
10X Ampligase Buffer |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00 |
2.00
|
Total |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00 |
10.00
|
strip 3
|
NTC:500
|
NTC:1000
|
BIS-DNA required (ng) |
5 |
5
|
JK # |
0 |
0
|
Conc. |
0 |
0
|
Volume (ul) |
0.00 |
0.00
|
H2O (ul) |
5.50 |
3.99
|
V1N3 amount (ng) |
1.10 |
2.20
|
V1N3 conc. (ng/ul) |
0.73 |
0.73
|
V1N3 volume (ul) |
1.50 |
3.01
|
10X Ampligase Buffer |
1.00 |
1.00
|
H2O in Mix |
2.00 |
2.00
|
Total |
10.00 |
10.00
|
V1N3 & Ampligase Buffer mix
Mix 0.11
|
1x
|
4x
|
Diluted V1N3 |
0.15 |
0.60
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.15 |
|
Mix 0.22
|
1x
|
6x
|
Diluted V1N3 |
0.30 |
1.80
|
10X Ampligase Buffer |
1.00 |
6.00
|
H2O |
2.00 |
12.00
|
Sum |
3.30 |
|
Mix 0.44
|
1x
|
4x
|
Diluted V1N3 |
0.60 |
2.40
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.60 |
|
Mix 0.55
|
1x
|
4x
|
Diluted V1N3 |
0.75 |
3.00
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
3.75 |
|
Mix 1.1
|
1x
|
6x
|
Diluted V1N3 |
1.50 |
9.00
|
10X Ampligase Buffer |
1.00 |
6.00
|
H2O |
2.00 |
12.00
|
Sum |
4.50 |
|
Mix 2.2
|
1x
|
4x
|
Diluted V1N3 |
3.01 |
12.04
|
10X Ampligase Buffer |
1.00 |
4.00
|
H2O |
2.00 |
8.00
|
Sum |
6.01 |
|
PAGE verification
- Loaded 4ul of PCR products in 6% TBE gel
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 500to1.jpg
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 1000to1.jpg