Noi/NOTES/2011-5-28

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Revision as of 21:51, 28 May 2011 by >Noi
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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
  1. calculate 1.5ug vol. of DNA sample, and the vol. of H2O to adjust total volume to 20ul
  2. add water to 96-conversion plate, then add DNA
  3. add 130ul of CT conversion reagent to 20ul of DNA sample, mix sample by pipetting up and down
  4. seal the plate with sticky film
  5. Transfer the plate to a thermal cycler and perform the following steps.
    1. 98C for 10mim
    2. 64C for 2.5hr
    3. 4 hold (can be up to 20hr)
  6. add 400ul of M-binding buffer to the well of the Silicon-A Binding plate mounted on a collection plate
  7. transfer the samples from the conversion plate to the well of the Silicon-A Binding plate, mix by pipetting up and down
  8. centrifuge at >3000xg for 5min, discard the flow through
  9. add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5 min
  10. add 200ul of M-Desulphonation buffer to each well, incubate at RT (20C-30C) for 20min
  11. centrifuge at >3000xg for 5min, discard the flow through
  12. add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5min, discard the flow through
  13. add another 400ul of M-Wash buffer, centrifuge for 10 min
  14. place the Silicon-A binding plate onto an Elution plate, add 15ul of M-Elution buffer directly to each well
  15. incubate for 5min, centrifuge at >3000xg for 3min to elute the DNA
  16. 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.


  • 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