Revision as of 20:59, 29 July 2013 by >Noi
DNA methylation assay for Blueprint project
Bisulfite conversion
I quantified gDNA concentration by Qubit dsDNA assay. Note that the volume of each sample they sent us was variied so I can not summarize total amount of each sample. However, based on concentration by Qubit the total amount we got of each sample less than 1ug as they claimed. Therefore I may not have enough gDNA for the second trial even I used only 500ng for bisulfite conversion. Also, there were few samples which total amount is very low so total amount for bis-cvt less than 300ng.
- I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs
|
WellIDs
|
Conc. (ng/ul)
|
Volume (ul)
|
H2O (ul)
|
CT conversion reagent (ul)
|
Total volume (ul)
|
Total amount (ng)
|
1 |
A1 |
37.71 |
13.26 |
6.74 |
130.00 |
150.00 |
500.00
|
2 |
B1 |
39.43 |
12.68 |
7.32 |
130.00 |
150.00 |
500.00
|
3 |
C1 |
50.57 |
9.89 |
10.11 |
130.00 |
150.00 |
500.00
|
4 |
D1 |
34.86 |
14.34 |
5.66 |
130.00 |
150.00 |
500.00
|
5 |
E1 |
56.00 |
8.93 |
11.07 |
130.00 |
150.00 |
500.00
|
6 |
F1 |
44.29 |
11.29 |
8.71 |
130.00 |
150.00 |
500.00
|
7 |
G1 |
73.71 |
6.78 |
13.22 |
130.00 |
150.00 |
500.00
|
8 |
H1 |
29.71 |
16.83 |
3.17 |
130.00 |
150.00 |
500.00
|
9 |
A2 |
40.29 |
12.41 |
7.59 |
130.00 |
150.00 |
500.00
|
10 |
B2 |
33.43 |
14.96 |
5.04 |
130.00 |
150.00 |
500.00
|
11 |
C2 |
44.86 |
11.15 |
8.85 |
130.00 |
150.00 |
500.00
|
12 |
D2 |
52.86 |
9.46 |
10.54 |
130.00 |
150.00 |
500.00
|
**13 |
E2 |
11.34 |
9.00 |
11.00 |
130.00 |
150.00 |
102.09
|
*14 |
F2 |
36.86 |
9.00 |
11.00 |
130.00 |
150.00 |
331.71
|
**15 |
G2 |
26.74 |
9.00 |
11.00 |
130.00 |
150.00 |
240.69
|
*16 |
H2 |
50.00 |
9.00 |
11.00 |
130.00 |
150.00 |
450.00
|
17 |
A3 |
34.00 |
14.71 |
5.29 |
130.00 |
150.00 |
500.00
|
18 |
B3 |
31.14 |
16.06 |
3.94 |
130.00 |
150.00 |
500.00
|
19 |
C3 |
26.46 |
18.90 |
1.10 |
130.00 |
150.00 |
500.00
|
*20 |
D3 |
20.29 |
20.00 |
0.00 |
130.00 |
150.00 |
405.71
|
*21 |
E3 |
18.23 |
20.00 |
0.00 |
130.00 |
150.00 |
364.57
|
22 |
F3 |
29.43 |
16.99 |
3.01 |
130.00 |
150.00 |
500.00
|
23 |
G3 |
47.71 |
10.48 |
9.52 |
130.00 |
150.00 |
500.00
|
24 |
H3 |
32.57 |
15.35 |
4.65 |
130.00 |
150.00 |
500.00
|
25 |
A4 |
29.14 |
17.16 |
2.84 |
130.00 |
150.00 |
500.00
|
*26 |
B4 |
19.60 |
20.00 |
0.00 |
130.00 |
150.00 |
392.00
|
27 |
C4 |
24.31 |
20.00 |
0.00 |
130.00 |
150.00 |
486.29
|
28 |
D4 |
23.66 |
20.00 |
0.00 |
130.00 |
150.00 |
473.14
|
29 |
E4 |
47.43 |
10.54 |
9.46 |
130.00 |
150.00 |
500.00
|
30 |
F4 |
76.86 |
6.51 |
13.49 |
130.00 |
150.00 |
500.00
|
31 |
G4 |
40.00 |
12.50 |
7.50 |
130.00 |
150.00 |
500.00
|
32 |
H4 |
32.29 |
15.49 |
4.51 |
130.00 |
150.00 |
500.00
|
33 |
A5 (12878) |
58.00 |
8.62 |
11.38 |
130.00 |
150.00 |
500.00
|
34 |
B5 (12878) |
59.00 |
8.47 |
11.53 |
131.00 |
151.00 |
500.00
|
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
- Eluted bisulfite converted DNA form the bead with 20ul H2O
- I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.
ssDNA Qubit assay result
Sample IDs
|
WellIDs
|
Start amount (ng)
|
Conc. (ng/ul)
|
Yield in 17ul (ng)
|
Yield left after Qubit (15ul)
|
% Recovery in 17ul
|
1 |
A1 |
500.00 |
21.00 |
357.00 |
315.00 |
71.40
|
2 |
B1 |
500.00 |
24.40 |
414.80 |
366.00 |
82.96
|
3 |
C1 |
500.00 |
19.10 |
324.70 |
286.50 |
64.94
|
4 |
D1 |
500.00 |
22.60 |
384.20 |
339.00 |
76.84
|
5 |
E1 |
500.00 |
20.60 |
350.20 |
309.00 |
70.04
|
6 |
F1 |
500.00 |
21.20 |
360.40 |
318.00 |
72.08
|
7 |
G1 |
500.00 |
22.00 |
374.00 |
330.00 |
74.80
|
8 |
H1 |
500.00 |
22.20 |
377.40 |
333.00 |
75.48
|
9 |
A2 |
500.00 |
20.60 |
350.20 |
309.00 |
70.04
|
10 |
B2 |
500.00 |
24.00 |
408.00 |
360.00 |
81.60
|
11 |
C2 |
500.00 |
19.06 |
324.02 |
285.90 |
64.80
|
12 |
D2 |
500.00 |
18.78 |
319.26 |
281.70 |
63.85
|
13 |
E2 |
102.09 |
2.06 |
35.02 |
30.90 |
34.30
|
14 |
F2 |
331.71 |
8.08 |
137.36 |
121.20 |
41.41
|
15 |
G2 |
240.69 |
6.50 |
110.50 |
97.50 |
45.91
|
16 |
H2 |
450.00 |
12.32 |
209.44 |
184.80 |
46.54
|
17 |
A3 |
500.00 |
11.56 |
196.52 |
173.40 |
39.30
|
18 |
B3 |
500.00 |
12.24 |
208.08 |
183.60 |
41.62
|
19 |
C3 |
500.00 |
11.44 |
194.48 |
171.60 |
38.90
|
20 |
D3 |
405.71 |
7.92 |
134.64 |
118.80 |
33.19
|
21 |
E3 |
364.57 |
8.10 |
137.70 |
121.50 |
37.77
|
22 |
F3 |
500.00 |
11.38 |
193.46 |
170.70 |
38.69
|
23 |
G3 |
500.00 |
13.96 |
237.32 |
209.40 |
47.46
|
24 |
H3 |
500.00 |
19.72 |
335.24 |
295.80 |
67.05
|
25 |
A4 |
500.00 |
21.20 |
360.40 |
318.00 |
72.08
|
26 |
B4 |
392.00 |
23.00 |
391.00 |
345.00 |
99.74
|
27 |
C4 |
486.29 |
23.80 |
404.60 |
357.00 |
83.20
|
28 |
D4 |
473.14 |
31.40 |
533.80 |
471.00 |
112.82
|
29 |
E4 |
500.00 |
20.80 |
353.60 |
312.00 |
70.72
|
30 |
F4 |
500.00 |
10.46 |
177.82 |
156.90 |
35.56
|
31 |
G4 |
500.00 |
47.00 |
799.00 |
705.00 |
159.80
|
32 |
H4 |
500.00 |
56.00 |
952.00 |
840.00 |
190.40
|
33 |
A5 (12878) |
500.00 |
24.00 |
408.00 |
360.00 |
81.60
|
34 |
B5 (12878) |
500.00 |
24.60 |
418.20 |
369.00 |
83.64
|
- There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
- Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.
BSPP capture set up
Original set
- Conc.: 25.4 ng/ul (by Qubit ssDNA assay)
Probe:target
|
500:1
|
'
|
Probe size |
12000 |
|
Template |
150 |
ng
|
Human gDNA MW |
1.9500E+12 |
g/mol
|
Human gDNA (150ng) |
7.6923E-20 |
mol (150ng/1.9500E+12 g/mol)
|
Probe required (500:1) |
3.8462E-17 |
mol (7.6923E-20 mol * 500)
|
Probe MW (12,000 probes, 110nt) |
4.2900E+08 |
g/mol (12000 probes * 110nt * 325Da/nt)
|
Amount probe required |
1.6500E-08 |
g (4.2900E+08 g/mol * 3.8462E-17 mol)
|
Amount probe required |
16.50 |
ng
|
Subset A
- Conc.: 17.6ng/ul (by Qubit ssDNA assay)
Probe:target
|
500:1
|
'
|
Probe size |
566 |
probes
|
Template |
150 |
ng
|
Human gDNA MW |
1.9500E+12 |
g/mol (3E+09 * 650Da/bp+157.9Da)
|
Human gDNA (200ng) |
7.6923E-20 |
mol (200ng/1.9500E+12 g/mol)
|
Probe required (500:1) |
3.8462E-17 |
mol (1.5385E-19mol * 500)
|
Probe MW (566 probes, 1100nt) |
2.0235E+07 |
g/mol (566 probes * 110nt * 325Da/nt)
|
Amount probe required |
7.7825E-10 |
g 2.0235E+07 g/mol * 3.8462E-17mol)
|
Amount probe required |
0.78 |
ng
|
- So for 1:5:5 (Original:A:B), amount probe required = 0.78ng * 5 = 8.89ng
Subset B
- Conc.: 57.9ng/ul (by Qubit ssDNA assay)
Probe:target
|
500:1
|
'
|
Probe size |
11397 |
probes
|
Template |
150 |
ng
|
Human gDNA MW |
1.9500E+12 |
g/mol (3E+09 * 650Da/bp+157.9Da)
|
Human gDNA (200ng) |
7.6923E-20 |
mol (150ng/1.9500E+12 g/mol)
|
Probe required (500:1) |
3.8462E-17 |
mol (1.5385E-19mol * 500)
|
Probe MW (11,397 probes, 1100nt) |
4.0744E+08 |
g/mol (11397 probes * 110nt * 325Da/nt)
|
Amount probe required |
1.5671E-08 |
g (4.0744E+08 g/mol * 3.8462E-17 mol)
|
Amount probe required |
15.67 |
ng
|
- So for 1:5:5 (Original:A:B), amount probe required = 15.67ng * 5 = 78.35ng
Summary of probes required for cpature
Probe set
|
Conc. (ng/ul)
|
Vmount required
|
Volume (ul)/samples
|
Volume for ~38x rxn
|
Original |
25.40 |
16.50 |
0.65 |
24.69
|
Subset A |
17.60 |
3.89 |
0.22 |
8.40
|
Subset B |
57.90 |
78.35 |
1.35 |
51.42
|
- I will do two captures for positive control (12878) to see the consistency between the two technical replicates and one NTC
- I will save at least 2ul of bis-cvt DNA for bisulfite PCR if we need to do that. However, Dr. Zhang suggested us to ignore the recommended_4 site.
Sample IDs
|
WellIDs
|
Conc. (ng/ul)
|
Volume required (ul)
|
BP-O (ul)
|
BP.norm-A (ul)
|
BP.norm-B (ul)
|
10X Ampligase Buffer (ul)
|
H2O (ul)
|
Total vulome (ul)
|
Total biscvt DNA (ng)
|
1 |
A1 |
21.00 |
7.14 |
0.65 |
0.22 |
1.35 |
2.00 |
8.64 |
20.00 |
150.00
|
2 |
B1 |
24.40 |
6.15 |
0.65 |
0.22 |
1.35 |
2.00 |
9.63 |
20.00 |
150.00
|
3 |
C1 |
19.10 |
7.85 |
0.65 |
0.22 |
1.35 |
2.00 |
7.93 |
20.00 |
150.00
|
4 |
D1 |
22.60 |
6.64 |
0.65 |
0.22 |
1.35 |
2.00 |
9.14 |
20.00 |
150.00
|
5 |
E1 |
20.60 |
7.28 |
0.65 |
0.22 |
1.35 |
2.00 |
8.50 |
20.00 |
150.00
|
6 |
F1 |
21.20 |
7.08 |
0.65 |
0.22 |
1.35 |
2.00 |
8.70 |
20.00 |
150.00
|
7 |
G1 |
22.00 |
6.82 |
0.65 |
0.22 |
1.35 |
2.00 |
8.96 |
20.00 |
150.00
|
8 |
H1 |
22.20 |
6.76 |
0.65 |
0.22 |
1.35 |
2.00 |
9.02 |
20.00 |
150.00
|
9 |
A2 |
20.60 |
7.28 |
0.65 |
0.22 |
1.35 |
2.00 |
8.50 |
20.00 |
150.00
|
10 |
B2 |
24.00 |
6.25 |
0.65 |
0.22 |
1.35 |
2.00 |
9.53 |
20.00 |
150.00
|
11 |
C2 |
19.06 |
7.87 |
0.65 |
0.22 |
1.35 |
2.00 |
7.91 |
20.00 |
150.00
|
12 |
D2 |
18.78 |
7.99 |
0.65 |
0.22 |
1.35 |
2.00 |
7.79 |
20.00 |
150.00
|
13 |
E2 |
2.06 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
26.78
|
14 |
F2 |
8.08 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
105.04
|
15 |
G2 |
6.50 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
84.50
|
16 |
H2 |
12.32 |
12.18 |
0.65 |
0.22 |
1.35 |
2.00 |
3.60 |
20.00 |
150.00
|
17 |
A3 |
11.56 |
12.98 |
0.65 |
0.22 |
1.35 |
2.00 |
2.80 |
20.00 |
150.00
|
18 |
B3 |
12.24 |
12.25 |
0.65 |
0.22 |
1.35 |
2.00 |
3.53 |
20.00 |
150.00
|
19 |
C3 |
11.44 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
148.72
|
20 |
D3 |
7.92 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
102.96
|
21 |
E3 |
8.10 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
105.30
|
22 |
F3 |
11.38 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
147.94
|
23 |
G3 |
13.96 |
10.74 |
0.65 |
0.22 |
1.35 |
2.00 |
5.04 |
20.00 |
150.00
|
24 |
H3 |
19.72 |
7.61 |
0.65 |
0.22 |
1.35 |
2.00 |
8.17 |
20.00 |
150.00
|
25 |
A4 |
21.20 |
7.08 |
0.65 |
0.22 |
1.35 |
2.00 |
8.70 |
20.00 |
150.00
|
26 |
B4 |
23.00 |
6.52 |
0.65 |
0.22 |
1.35 |
2.00 |
9.26 |
20.00 |
150.00
|
27 |
C4 |
23.80 |
6.30 |
0.65 |
0.22 |
1.35 |
2.00 |
9.48 |
20.00 |
150.00
|
28 |
D4 |
31.40 |
4.78 |
0.65 |
0.22 |
1.35 |
2.00 |
11.00 |
20.00 |
150.00
|
29 |
E4 |
20.80 |
7.21 |
0.65 |
0.22 |
1.35 |
2.00 |
8.57 |
20.00 |
150.00
|
30 |
F4 |
10.46 |
13.00 |
0.65 |
0.22 |
1.35 |
2.00 |
2.78 |
20.00 |
135.98
|
31 |
G4 |
47.00 |
3.19 |
0.65 |
0.22 |
1.35 |
2.00 |
12.59 |
20.00 |
150.00
|
32 |
H4 |
56.00 |
2.68 |
0.65 |
0.22 |
1.35 |
2.00 |
13.10 |
20.00 |
150.00
|
33 |
A5 (12878) |
24.00 |
6.25 |
0.65 |
0.22 |
1.35 |
2.00 |
9.53 |
20.00 |
150.00
|
34 |
B5 (12878) |
24.60 |
6.10 |
0.65 |
0.22 |
1.35 |
2.00 |
9.68 |
20.00 |
150.00
|
35 |
C5 (NTC) |
0.00 |
0.00 |
0.65 |
0.22 |
1.35 |
2.00 |
15.78 |
20.00 |
0.00
|
Reaction mix
Components
|
1x rxn
|
38 rxn mix
|
10X Ampligase buffer |
2.00 |
76.00
|
BP-O |
0.65 |
24.70
|
BP.norm-A |
0.22 |
8.36
|
BP.norm-B |
1.35 |
51.30
|
Total |
4.22 |
160.36
|
- Aliquot ~18ul to 8-tube strip and add 4.22ul of reaction mix to the 96-well plate with bis-cvt DNA and H2O with multichannel pipette
- Mix 15 time by pipetting
- Add 40ul of mineral oil & seal with silicone sealing mat
- Spin the plate and place on thermocycler
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2ul SLN mix (10x Titanium Taq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 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 volume (ul), total 80ul
|
Titanium Taq (Klentaq fragment) |
10 |
U/ul |
2 |
U/ul |
16.00
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
8.00
|
dNTP |
1 |
mM |
100 |
uM |
8.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
8.00
|
H2O |
|
|
|
|
40.00
|
- Aliquot 9ul to 8-tube strip and add 2ul to each well with multichannel pipette.
- Note: Actually, Clontech doesn't provide the concentration of Titanium Taq. They only said 50X concentration. I assume it has the same concentration as Stoffel fragment.
PCR Amplification (TEST)
- Before amplify with barcode primer I test if the capture work by doing PCR in small volume (25ul) with 2ul of circularized DNA
Components
|
1x rxn
|
30 x rxn mix
|
Captured template |
2.00 |
0.00
|
10uM AmpF6.4Sol |
0.50 |
15.00
|
10uM AmpR6.3.Ind1 |
0.50 |
15.00
|
2X KAPA SYBR MM |
12.50 |
375.00
|
H2O |
9.50 |
285.00
|
Total |
25.00 |
|
- Aliquot 23ul, add 2ul of circularized DNA template
Program (Eppendorf Realplex
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Loaded 3ul of PCR product in TBE gel
File:ZhangLab 2 2013-07-22 19hr 23min TEST-BP-REAL.jpg
- It seem like I din't see very clear expected bands between 400-480bp, and the background is very high. I wonder if if added too much template, but for previous experiment I add 10ul of template in total 50ul, and got very clear bands.
- I TEST PCR again by varying template volume (0.5, 1, 2, and 4ul in total 25ul) to test if the volume of template interfere PCR.
- From the result, it seem like smaller volume of template show less background and stronger expected products. However, it didn't look as clean as I got from the first test experiment. I suspect that Exo I/III may not completely digest ds/ssDNA. I then tested enzyme activity by incubating equal amount of probes and bis-cvt gDNA I used in capture and I saw very clean gel. This suggested that enzymatic activity was good. I also run check circularized template in TBU gel (loaded 3ul) to see if there is the probe or gDNA left. I did not see either. However by PAGE verification, I may not be able to see the left over of probes and gDNA if the level was very low.
File:ZhangLab 2 2013-07-22 15hr 11min confirmExoI-III-digestedprobes.jpg File:ZhangLab 2 2013-07-23 12hr 59min TBU circularized DNA.jpg
Gel1 = ExoI/III with equal amount of probe and bis-cvt gDNA (loaded difeerent amount)
Gel2 = 3ul of NTC and well B5 circularized DNA
- I decided to used 4% of template and perform PCR amplification in total 100ul reaction to make sequencing libraries.
Components
|
1x rxn
|
38 rxn mix
|
Captured template |
4.00 |
0.00
|
10uM AmpF6.4Sol |
2.00 |
76.00
|
10uM AmpR6.3Sol |
2.00 |
0.00
|
2X KAPA SYBR MM |
50.00 |
1900.00
|
H2O |
42.00 |
1596.00
|
Total |
100.00 |
|
Program (Eppendorf Realplex
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Purified PCR product with 0.7 volume Ampure beads (I dilute bead 1:1 with 20% PEG/5M NaCl before adding 0.7 volume for purification).
- Eluted with 30ul H2O
- Performed PAGE quantification by loading sample 2ul
File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A1-H1.jpg File:ZhangLab 2 2013-07-23 17hr 11min qPCR BP-REAL A2-H2.jpg
File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A3-H3.jpg File:ZhangLab 2 2013-07-23 17hr 59min qPCR BP-REAL A4-B5.jpg
PAGE quantification
Well IDs
|
Index
|
Conc. (ng/ul)
|
Yields in 30ul (ng)
|
Well IDs
|
Pool1 volume (ul)
|
Amount
|
Pool2 volume (ul)
|
Amount (ng)
|
A1 |
Index 57 |
6.17 |
185.16 |
A1 |
8.10 |
50.00 |
NA |
NA
|
B1 |
Index 58 |
6.21 |
186.45 |
B1 |
8.05 |
50.00 |
NA |
NA
|
C1 |
Index 59 |
6.42 |
192.60 |
C1 |
7.79 |
50.00 |
NA |
NA
|
D1 |
Index 60 |
6.20 |
186.10 |
D1 |
8.06 |
50.00 |
NA |
NA
|
E1 |
Index 61 |
6.32 |
189.72 |
E1 |
7.91 |
50.00 |
NA |
NA
|
F1 |
Index 62 |
6.61 |
198.32 |
F1 |
7.56 |
50.00 |
NA |
NA
|
G1 |
Index 63 |
6.65 |
199.44 |
G1 |
7.52 |
50.00 |
NA |
NA
|
H1 |
Index 64 |
5.93 |
177.79 |
H1 |
8.44 |
50.00 |
NA |
NA
|
A2 |
Index 65 |
4.94 |
148.24 |
A2 |
10.12 |
50.00 |
NA |
NA
|
B2 |
Index 66 |
5.54 |
166.12 |
B2 |
9.03 |
50.00 |
NA |
NA
|
C2 |
Index 67 |
4.93 |
147.94 |
C2 |
10.14 |
50.00 |
NA |
NA
|
D2 |
Index 68 |
5.56 |
166.68 |
D2 |
9.00 |
50.00 |
NA |
NA
|
E2 |
Index 69 |
4.53 |
135.81 |
E2 |
11.04 |
50.00 |
NA |
NA
|
F2 |
Index 70 |
5.39 |
161.60 |
F2 |
9.28 |
50.00 |
NA |
NA
|
G2 |
Index 71 |
4.99 |
149.63 |
G2 |
10.02 |
50.00 |
NA |
NA
|
H2 |
Index 72 |
5.61 |
168.28 |
H2 |
8.91 |
50.00 |
NA |
NA
|
A3 |
Index 73 |
1.47 |
44.22 |
A3 |
NA |
NA |
12.21 |
18.00
|
B3 |
Index 74 |
2.11 |
63.34 |
B3 |
23.68 |
50.00 |
NA |
NA
|
C3 |
Index 75 |
2.81 |
84.45 |
C3 |
17.76 |
50.00 |
NA |
NA
|
D3 |
Index 76 |
3.68 |
110.48 |
D3 |
13.58 |
50.00 |
NA |
NA
|
E3 |
Index 77 |
3.92 |
117.57 |
E3 |
12.76 |
50.00 |
NA |
NA
|
F3 |
Index 78 |
3.61 |
108.40 |
F3 |
13.84 |
50.00 |
NA |
NA
|
G3 |
Index 79 |
3.68 |
110.29 |
G3 |
13.60 |
50.00 |
NA |
NA
|
H3 |
Index 80 |
3.67 |
110.14 |
H3 |
13.62 |
50.00 |
NA |
NA
|
A4 |
Index 81 |
0.62 |
18.67 |
A4 |
NA |
NA |
28.93 |
18.00
|
B4 |
Index 82 |
5.31 |
159.39 |
B4 |
9.41 |
50.00 |
NA |
NA
|
C4 |
Index 83 |
5.10 |
153.05 |
C4 |
9.80 |
50.00 |
NA |
NA
|
D4 |
Index 84 |
5.88 |
176.34 |
D4 |
8.51 |
50.00 |
NA |
NA
|
E4 |
Index 85 |
5.85 |
175.36 |
E4 |
8.55 |
50.00 |
NA |
NA
|
F4 |
Index 86 |
5.89 |
176.80 |
F4 |
8.48 |
50.00 |
NA |
NA
|
G4 |
Index 87 |
5.89 |
176.78 |
G4 |
8.49 |
50.00 |
NA |
NA
|
H4 |
Index 88 |
5.46 |
163.76 |
H4 |
9.16 |
50.00 |
NA |
NA
|
A5 |
Index 89 |
5.24 |
157.26 |
A5 |
9.54 |
50.00 |
NA |
NA
|
B5 |
Index 90 |
4.52 |
135.63 |
B5 |
11.06 |
50.00 |
NA |
NA
|
- There were two samples have very low concentration, so I pool these two libraries together and purified in on lane of 5-well TBE gel
- The rest of libraries were pooled together and purified in 2 of 5-well TBE gels.
PAGE size-selection
File:ZhangLab 2 2013-07-23 21hr 32min BP-SeqLib-PAGE-SS.jpg File:ZhangLab 2 2013-07-23 21hr 36min BP-SeqLib-PAGE-SS DD-bisPCR.jpg
- Note: I also perforedm PAGE-size selection of bis PCR performed by Dinh by pooling PCR products normalized based on qt values of qPCR. After PAGE size selection, the libraries were resuspended in 12ul (Library ID: DD-bisPCR-BPrecommended4-Jul19, Index 9-20, 23-44)