Revision as of 20:54, 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