Noi/NOTES/2011-5-28: Difference between revisions
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* Actual speed for centrifugation: 2,000rpm. | * Actual speed for centrifugation: 2,000rpm. | ||
*Note: The ID used for HIV samples will correspond to the position in the 96-well plate. | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Order''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID in Zhang lab''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''volume for 1200ng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''H20 (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total amount for bis-cvt (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | |||
|- | |||
| 1||A1||100||12||8||1200||130||150 | |||
|- | |||
| 2||A2||100||12||8||1200||130||150 | |||
|- | |||
| 3||A3||100||12||8||1200||130||150 | |||
|- | |||
| 4||A4||100||12||8||1200||130||150 | |||
|- | |||
| 5||A5||100||12||8||1200||130||150 | |||
|- | |||
| 6||A6||100||12||8||1200||130||150 | |||
|- | |||
| 7||A7||100||12||8||1200||130||150 | |||
|- | |||
| 8||A8||100||12||8||1200||130||150 | |||
|- | |||
| 9||A9||100||12||8||1200||130||150 | |||
|- | |||
| 10||A10||100||12||8||1200||130||150 | |||
|- | |||
| 11||A11||100||12||8||1200||130||150 | |||
|- | |||
| 12||A12||100||12||8||1200||130||150 | |||
|- | |||
| 13||B1||100||12||8||1200||130||150 | |||
|- | |||
| 14||B2||100||12||8||1200||130||150 | |||
|- | |||
| 15||B3||100||12||8||1200||130||150 | |||
|- | |||
| 16||B4||100||12||8||1200||130||150 | |||
|- | |||
| 17||B5||100||12||8||1200||130||150 | |||
|- | |||
| 18||B6||100||12||8||1200||130||150 | |||
|- | |||
| 19||B7||100||12||8||1200||130||150 | |||
|- | |||
| 20||B8||100||12||8||1200||130||150 | |||
|- | |||
| 21||B9||100||12||8||1200||130||150 | |||
|- | |||
| 22||B10||100||12||8||1200||130||150 | |||
|- | |||
| 23||B11||100||12||8||1200||130||150 | |||
|- | |||
| 24||B12||100||12||8||1200||130||150 | |||
|- | |||
| 25||C1||100||12||8||1200||130||150 | |||
|- | |||
| 26||C2||100||12||8||1200||130||150 | |||
|- | |||
| 27||C3||100||12||8||1200||130||150 | |||
|- | |||
| 28||C4||100||12||8||1200||130||150 | |||
|- | |||
| 29||C5||100||12||8||1200||130||150 | |||
|- | |||
| 30||C6||100||12||8||1200||130||150 | |||
|- | |||
| 31||C7||100||12||8||1200||130||150 | |||
|- | |||
| 32||C8||100||12||8||1200||130||150 | |||
|- | |||
| 33||C9||100||12||8||1200||130||150 | |||
|- | |||
| 34||C10||100||12||8||1200||130||150 | |||
|- | |||
| 35||C11||100||12||8||1200||130||150 | |||
|- | |||
| 36||C12||100||12||8||1200||130||150 | |||
|- | |||
| 37||D1||100||12||8||1200||130||150 | |||
|- | |||
| 38||D2||100||12||8||1200||130||150 | |||
|- | |||
| 39||D3||100||12||8||1200||130||150 | |||
|- | |||
| 40||N37-1||100||12||8||1200||130||150 | |||
|- | |||
| 41||N37-2||100||12||8||1200||130||150 | |||
|- | |||
| 42||N37-3||100||12||8||1200||130||150 | |||
|- | |||
| 43||N37-4||100||12||8||1200||130||150 | |||
|- | |||
| 44||N37-5||100||12||8||1200||130||150 | |||
|- | |||
| 45||N37-6||100||12||8||1200||130||150 | |||
|- | |||
| 46||N37-7||100||12||8||1200||130||150 | |||
|- | |||
| 47||N37-8||100||12||8||1200||130||150 | |||
|- | |||
| 48||N37-9||100||12||8||1200||130||150 | |||
|- | |||
| 49||N37-10||100||12||8||1200||130||150 | |||
|}<br> | |||
* Plate layout | * Plate layout |
Revision as of 00:59, 29 May 2011
BSPP DMR330k capture of HIV patients samples and N37
- May 19, 2011, received 39 gDNA from the blood samples of HIV patients form Tropher's lab, Medical School, UCSD
- Ma7 25, 2011, received 10 gDNA from all tissues of N37 donor from Jin Billy Li, Standford Univ
Sample ID | PID | New order in Zhang lab | Place in 96plate | Volume (ul) | Conc. (ng/ul) | Total amount (ug) |
3 | 26406 102 | 1 | A1 | 21 | 100 | 2.1 |
4 | 26406 108 | 2 | A2 | 21 | 100 | 2.1 |
5 | 26604 054 | 3 | A3 | 21 | 100 | 2.1 |
6 | 26604 066 | 4 | A4 | 21 | 100 | 2.1 |
11 | NW578 000 | 5 | A5 | 21 | 100 | 2.1 |
12 | NW578 012 | 6 | A6 | 21 | 100 | 2.1 |
13 | 58346 006 | 7 | A7 | 21 | 100 | 2.1 |
14 | 58346 012 | 8 | A8 | 21 | 100 | 2.1 |
15 | CA198 006 | 9 | A9 | 21 | 100 | 2.1 |
16 | CA198 C12 | 10 | A10 | 21 | 100 | 2.1 |
17 | CA219 000 | 11 | A11 | 21 | 100 | 2.1 |
18 | CA219 006 | 12 | A12 | 21 | 100 | 2.1 |
19 | CA275 000 | 13 | B1 | 21 | 100 | 2.1 |
20 | CA275 006 | 14 | B2 | 21 | 100 | 2.1 |
21 | CB252 000 | 15 | B3 | 21 | 100 | 2.1 |
22 | CB252 006 | 16 | B4 | 21 | 100 | 2.1 |
24 | CC182 012 | 17 | B5 | 21 | 100 | 2.1 |
25 | 24371 000 | 18 | B6 | 21 | 100 | 2.1 |
26 | 24371 012 | 19 | B7 | 21 | 100 | 2.1 |
27 | NW470 012 | 20 | B8 | 21 | 100 | 2.1 |
28 | NW470 024 | 21 | B9 | 21 | 100 | 2.1 |
30 | 27054 012 | 22 | B10 | 21 | 100 | 2.1 |
31 | CA239 000 | 23 | B11 | 21 | 100 | 2.1 |
33 | CE214 000 | 24 | B12 | 21 | 100 | 2.1 |
34 | CE214 006 | 25 | C1 | 21 | 100 | 2.1 |
35 | CB255 000 | 26 | C2 | 21 | 100 | 2.1 |
36 | CB255 006 | 27 | C3 | 21 | 100 | 2.1 |
37 | 21003 000 | 28 | C4 | 21 | 100 | 2.1 |
38 | 21003 012 | 29 | C5 | 21 | 100 | 2.1 |
39 | CG139 006 | 30 | C6 | 21 | 100 | 2.1 |
40 | CG139 018 | 31 | C7 | 21 | 100 | 2.1 |
41 | NM633 012 | 32 | C8 | 21 | 100 | 2.1 |
42 | NM633 024 | 33 | C9 | 21 | 100 | 2.1 |
43 | NM805 012 | 34 | C10 | 21 | 100 | 2.1 |
44 | NM805 024 | 35 | C11 | 21 | 100 | 2.1 |
45 | CB199 000 | 36 | C12 | 21 | 100 | 2.1 |
46 | 26592 060 | 37 | D1 | 21 | 100 | 2.1 |
47 | 24054 030 | 38 | D2 | 21 | 100 | 2.1 |
48 | 24054 036 | 39 | D3 | 21 | 100 | 2.1 |
Number | Sample | Concentration (ug/ul) | Volume (ul) | Total amount (ug) | NewID | TE buffer (ul) | Total volume (ul) | Final conc. (ng/ul) |
1.00 | Cerebellum | 1.38 | 1.45 | 2.00 | N37-1 | 18.55 | 20.00 | 100.05 |
2.00 | Colon | 1.44 | 1.39 | 2.00 | N37-2 | 18.61 | 20.00 | 100.08 |
3.00 | Frontal lobe | 0.91 | 2.20 | 2.00 | N37-3 | 17.80 | 20.00 | 100.10 |
4.00 | Heart | 0.73 | 2.74 | 2.00 | N37-4 | 17.26 | 20.00 | 100.01 |
5.00 | Small intestine | 1.09 | 1.83 | 1.99 | N37-5 | 18.17 | 20.00 | 99.74 |
6.00 | Liver | 0.93 | 2.15 | 2.00 | N37-6 | 17.85 | 20.00 | 99.98 |
7.00 | Lung | 0.50 | 4.00 | 2.00 | N37-7 | 16.00 | 20.00 | 100.00 |
8.00 | Skeletal muscle | 0.57 | 3.51 | 2.00 | N37-8 | 16.49 | 20.00 | 100.04 |
9.00 | Pancrease | 1.48 | 1.35 | 2.00 | N37-9 | 18.65 | 20.00 | 99.90 |
10.00 | Stomach | 1.17 | 1.71 | 2.00 | N37-10 | 18.29 | 20.00 | 100.04 |
- Note: For N37 sample set, TE buffer was added to bring up volume to 20ul and adjusted the final concentration to ~100ng/ul
Bisulfite conversion
- EZ-96 DNA Methylation-Gold Kit
- Catalog# D5007 (Shallow-Well Format)
- Reagent list
- CT conversion reagent: to the CT conversion reagent bottle, add 9mL H2O, 500ul M-dissolving buffer, and 3mL M-dilution buffer, mix at RT with frequent vortexing or shaking for 15min
- M-Binding buffer
- M-Wash buffer: add 144mL of 100% EtOH to the 36mL M-Wash buffer (total volume = 180mL)
- M-desulphonation buffer
- Procedure
- calculate 1.2ug vol. of DNA sample, and the vol. of H2O to adjust total volume to 20ul
- add water to 96-conversion plate, then add DNA
- add 130ul of CT conversion reagent to 20ul of DNA sample, mix sample by pipetting up and down
- seal the plate with sticky film
- Transfer the plate to a thermal cycler and perform the following steps.
- 98C for 10mim
- 64C for 2.5hr
- 4 hold (can be up to 20hr)
- add 400ul of M-binding buffer to the well of the Silicon-A Binding plate mounted on a collection plate
- transfer the samples from the conversion plate to the well of the Silicon-A Binding plate, mix by pipetting up and down
- centrifuge at >3000xg for 5min, discard the flow through
- add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5 min
- add 200ul of M-Desulphonation buffer to each well, incubate at RT (20C-30C) for 20min
- centrifuge at >3000xg for 5min, discard the flow through
- add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5min, discard the flow through
- add another 400ul of M-Wash buffer, centrifuge for 10 min
- place the Silicon-A binding plate onto an Elution plate, add 15ul of M-Elution buffer directly to each well
- incubate for 5min, centrifuge at >3000xg for 3min to elute the DNA
- DNA samples are ready for the next analysis or store at -20C for later use
- Note: for long term storage, store at or below -70C
- Actual speed for centrifugation: 2,000rpm.
- Note: The ID used for HIV samples will correspond to the position in the 96-well plate.
Order | Sample ID in Zhang lab | Conc. (ng/ul) | volume for 1200ng (ul) | H20 (ul) | Total amount for bis-cvt (ng) | CT conversion reagent (ul) | Total volume (ul) |
1 | A1 | 100 | 12 | 8 | 1200 | 130 | 150 |
2 | A2 | 100 | 12 | 8 | 1200 | 130 | 150 |
3 | A3 | 100 | 12 | 8 | 1200 | 130 | 150 |
4 | A4 | 100 | 12 | 8 | 1200 | 130 | 150 |
5 | A5 | 100 | 12 | 8 | 1200 | 130 | 150 |
6 | A6 | 100 | 12 | 8 | 1200 | 130 | 150 |
7 | A7 | 100 | 12 | 8 | 1200 | 130 | 150 |
8 | A8 | 100 | 12 | 8 | 1200 | 130 | 150 |
9 | A9 | 100 | 12 | 8 | 1200 | 130 | 150 |
10 | A10 | 100 | 12 | 8 | 1200 | 130 | 150 |
11 | A11 | 100 | 12 | 8 | 1200 | 130 | 150 |
12 | A12 | 100 | 12 | 8 | 1200 | 130 | 150 |
13 | B1 | 100 | 12 | 8 | 1200 | 130 | 150 |
14 | B2 | 100 | 12 | 8 | 1200 | 130 | 150 |
15 | B3 | 100 | 12 | 8 | 1200 | 130 | 150 |
16 | B4 | 100 | 12 | 8 | 1200 | 130 | 150 |
17 | B5 | 100 | 12 | 8 | 1200 | 130 | 150 |
18 | B6 | 100 | 12 | 8 | 1200 | 130 | 150 |
19 | B7 | 100 | 12 | 8 | 1200 | 130 | 150 |
20 | B8 | 100 | 12 | 8 | 1200 | 130 | 150 |
21 | B9 | 100 | 12 | 8 | 1200 | 130 | 150 |
22 | B10 | 100 | 12 | 8 | 1200 | 130 | 150 |
23 | B11 | 100 | 12 | 8 | 1200 | 130 | 150 |
24 | B12 | 100 | 12 | 8 | 1200 | 130 | 150 |
25 | C1 | 100 | 12 | 8 | 1200 | 130 | 150 |
26 | C2 | 100 | 12 | 8 | 1200 | 130 | 150 |
27 | C3 | 100 | 12 | 8 | 1200 | 130 | 150 |
28 | C4 | 100 | 12 | 8 | 1200 | 130 | 150 |
29 | C5 | 100 | 12 | 8 | 1200 | 130 | 150 |
30 | C6 | 100 | 12 | 8 | 1200 | 130 | 150 |
31 | C7 | 100 | 12 | 8 | 1200 | 130 | 150 |
32 | C8 | 100 | 12 | 8 | 1200 | 130 | 150 |
33 | C9 | 100 | 12 | 8 | 1200 | 130 | 150 |
34 | C10 | 100 | 12 | 8 | 1200 | 130 | 150 |
35 | C11 | 100 | 12 | 8 | 1200 | 130 | 150 |
36 | C12 | 100 | 12 | 8 | 1200 | 130 | 150 |
37 | D1 | 100 | 12 | 8 | 1200 | 130 | 150 |
38 | D2 | 100 | 12 | 8 | 1200 | 130 | 150 |
39 | D3 | 100 | 12 | 8 | 1200 | 130 | 150 |
40 | N37-1 | 100 | 12 | 8 | 1200 | 130 | 150 |
41 | N37-2 | 100 | 12 | 8 | 1200 | 130 | 150 |
42 | N37-3 | 100 | 12 | 8 | 1200 | 130 | 150 |
43 | N37-4 | 100 | 12 | 8 | 1200 | 130 | 150 |
44 | N37-5 | 100 | 12 | 8 | 1200 | 130 | 150 |
45 | N37-6 | 100 | 12 | 8 | 1200 | 130 | 150 |
46 | N37-7 | 100 | 12 | 8 | 1200 | 130 | 150 |
47 | N37-8 | 100 | 12 | 8 | 1200 | 130 | 150 |
48 | N37-9 | 100 | 12 | 8 | 1200 | 130 | 150 |
49 | N37-10 | 100 | 12 | 8 | 1200 | 130 | 150 |
- Plate layout
Plat layout | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A A | A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | A11 | A12 |
B | ||||||||||||
C B | B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
D | ||||||||||||
E C | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
F | ||||||||||||
G D | D1 | D2 | D3 | |||||||||
H | N37-1 | N37-2 | N37-3 | N37-4 | N37-5 | N37-6 | N37-7 | N37-8 | N37-9 | N37-10 |