Revision as of 23:37, 29 August 2012 by >Noi
Bisulfite conversion[edit]
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
|
- Note: Sample GL-5, GL-7 and GL-8 have lower yield than other samples, so I did bis-cvt in duplicate
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
- Elute with 30ul Elution buffer, after Qubit quantification I have ~25ul for BSPP capture
330k BSPP capture set up[edit]
Probe calculation[edit]
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
Reaction mix[edit]
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[edit]
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
2012.08.25
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind9-29)[edit]
- I tested if the capture works well by performing qPCR of few samples in small volume 25ul
- Sample te be tested: NTC, GL7,GL8 AL-1 and AL3
- Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction)
Components
|
1 rxn
|
5.2x rxn mix
|
Circularized DNA template |
5.00 |
0.00
|
10uM AmpF6.4Sol |
0.50 |
2.60
|
10uM AmpR6.3Sol |
0.50 |
2.60
|
50x SYBG |
0.20 |
1.04
|
2x Phusion MM |
12.50 |
65.00
|
H2O |
6.30 |
32.76
|
Total |
25.00 |
104.00
|
- Aliquot 20ul, add 5ul of DNA template
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:20120825 testqPCR BSPP-GL-AL.png
Components
|
1 rxn
|
45x rxn mix
|
Captured template |
20.00 |
0.00
|
10uM AmpF6.4Sol |
2.00 |
90.00
|
10uM AmpR6.3Indx (X=9-29) |
2.00 |
0.00
|
50x SYBG |
0.80 |
36.00
|
2x Phusion MM |
50.00 |
2250.00
|
H2O |
25.20 |
1134.00
|
Total |
100.00 |
3510.00
|
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min
(All reactions were stopped at cycle 19)
File:20120825 qPCR BSPP-GL-AL.png
Sample IDs
|
Indx
|
Sample IDs
|
Indx
|
Sample IDs
|
Indx
|
GL-1 |
AmpR6.3Indx9 |
GL-9 |
AmpR6.3Indx17 |
AL-1 |
AmpR6.3Indx25
|
GL-2 |
AmpR6.3Indx10 |
GL-10 |
AmpR6.3Indx18 |
AL-2 |
AmpR6.3Indx26
|
GL-3 |
AmpR6.3Indx11 |
GL-11 |
AmpR6.3Indx19 |
AL-3 |
AmpR6.3Indx27
|
GL-4 |
AmpR6.3Indx12 |
GL-12 |
AmpR6.3Indx20 |
AL-4 |
AmpR6.3Indx28
|
GL-5 |
AmpR6.3Indx13 |
GL-13 |
AmpR6.3Indx21 |
AL-5 |
AmpR6.3Indx29
|
GL-6 |
AmpR6.3Indx14 |
GL-14 |
AmpR6.3Indx22 |
|
|
GL-7 |
AmpR6.3Indx15 |
GL-15 |
AmpR6.3Indx23 |
|
|
GL-8 |
AmpR6.3Indx16 |
GL-16 |
AmpR6.3Indx24 |
|
|
- Took 80ul of PCR product from replicate1 and 2 (total volume 160ul)
- Purified with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl)
- Eluted with 60ul EB buffer
- Performed PAGE quantification by loading sample 1ul
File:ZhangLab 2 2012-08-25 21hr 25min PQ GL-1.jpg File:ZhangLab 2 2012-08-25 21hr 25min PQ GL-2.jpg
Sample IDs
|
Conc. (ng/ul)
|
Yields in 60ul (ng)
|
Volume for 200ng (ul)
|
GL-1 |
10.72 |
642.91 |
18.67
|
GL-2 |
12.24 |
734.53 |
16.34
|
GL-3 |
11.98 |
718.56 |
16.70
|
GL-4 |
12.17 |
730.11 |
16.44
|
GL-5 |
11.07 |
664.39 |
18.06
|
GL-6 |
12.74 |
764.43 |
15.70
|
GL-7 |
10.40 |
623.89 |
19.23
|
GL-8 |
9.74 |
584.46 |
20.53
|
GL-9 |
10.88 |
652.62 |
18.39
|
GL-10 |
11.16 |
669.83 |
17.92
|
GL-11 |
8.30 |
497.79 |
24.11
|
GL-12 |
9.40 |
564.06 |
21.27
|
GL-13 |
9.26 |
555.74 |
21.59
|
GL-14 |
7.18 |
430.56 |
27.87
|
GL-15 |
7.37 |
441.94 |
27.15
|
GL-16 |
8.25 |
494.83 |
24.25
|
AL-1 |
5.11 |
306.64 |
39.13
|
AL-2 |
6.38 |
382.88 |
31.34
|
AL-3 |
5.96 |
357.37 |
33.58
|
AL-4 |
4.91 |
294.31 |
40.77
|
AL-5 |
4.45 |
266.78 |
44.98
|
- Combine 200ng of each library.
-Perform PAGE size selection in 6% TBE 5-well gel (3 gels)
File:ZhangLab 2 2012-08-26 18hr 46min PAGE SS-1.jpg File:ZhangLab 2 2012-08-26 18hr 29min PAGE SS-2.jpg
* From gel images, DNA was overoaded
- Resuspended with H2O, total volume 48ul
File:ZhangLab 2 2012-08-26 22hr 24min PQ NP BSPP GL-AL.Ind9-29.jpg
dsDNA Qubit HS quantification[edit]
Library IDs
|
Concentration in the Qubit
|
Unit
|
uL used
|
Dilution
|
Final conc. (ng/ul)
|
Ave. Conc. (ng/ul)
|
AL-exome_Aug21 1ul |
46.60 |
ng/mL |
1.00 |
200 |
9.32 |
9.76
|
AL-exome_Aug21 2ul |
102.00 |
ng/mL |
2.00 |
100 |
10.20 |
|
NP_WGBS_N37_Aug26 1ul |
18.70 |
ng/mL |
1.00 |
200 |
3.74 |
3.80
|
NP_WGBS_N37_Aug26 2ul |
38.50 |
ng/mL |
2.00 |
100 |
3.85 |
|
NP-BSPP-GL-AL_Ind9-29_Aug23 1ul |
72.50 |
ng/mL |
1.00 |
200 |
14.50 |
14.95
|
NP-BSPP-GL-AL_Ind9-29_Aug23 2ul |
154.00 |
ng/mL |
2.00 |
100 |
15.40 |
|
Library IDs
|
Ave. Conc. (ng/ul)
|
Size (bp)
|
Conc. (nM)
|
MW (g/mole)
|
AL-exome_Aug21 |
9.76 |
300 |
53.52 |
182,377.9
|
NP_WGBS_N37_Aug26 |
3.80 |
500 |
12.49 |
303,857.9
|
NP-BSPP-GL-AL_Ind9-29_Aug23 |
14.95 |
375 |
65.59 |
227,932.9
|
- Quantified concentration by Qubit dsDNA assay HS (based on size 375bp): 14.95ng/ul or 65.59nM
- Concentration by PAGE quantification is 26.88ng/ul
- Library IDs: NP-BSPP-GL-AL_Ind9-29_Aug23, 2012, sequenced on HiSeqAug28 run at Biogem (Lane : 7-8)