Noi/NOTES/2011-5-28
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BSPP DMR330k capture of HIV patient samples and N37[edit]
- 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[edit]
- 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 |
- 2011_06_01
Qubit ss-DNA assay[edit]
- added 1ul of bis-cvt gDNA in total volume 200ul, so dilution factor = 200)
sample ID | Conc. In Qubit (ng/mL) | Final conc. (ng/ul) | Volume (ul) | Yields (ng) |
A1 | 184.00 | 36.80 | 15.00 | 552.00 |
A2 | 316.00 | 63.20 | 15.00 | 948.00 |
A3 | 208.00 | 41.60 | 15.00 | 624.00 |
A4 | 224.00 | 44.80 | 15.00 | 672.00 |
A5 | 176.00 | 35.20 | 15.00 | 528.00 |
A6 | 218.00 | 43.60 | 15.00 | 654.00 |
A7 | 240.00 | 48.00 | 15.00 | 720.00 |
A8 | 220.00 | 44.00 | 15.00 | 660.00 |
A9 | 365.00 | 73.00 | 15.00 | 1095.00 |
A10 | 220.00 | 44.00 | 15.00 | 660.00 |
A11 | 216.00 | 43.20 | 15.00 | 648.00 |
A12 | 285.00 | 57.00 | 15.00 | 855.00 |
B1 | 191.00 | 38.20 | 15.00 | 573.00 |
B2 | 263.00 | 52.60 | 15.00 | 789.00 |
B3 | 198.00 | 39.60 | 15.00 | 594.00 |
B4 | 153.00 | 30.60 | 15.00 | 459.00 |
B5 | 234.00 | 46.80 | 15.00 | 702.00 |
B6 | 301.00 | 60.20 | 15.00 | 903.00 |
B7 | 148.00 | 29.60 | 15.00 | 444.00 |
B8 | 209.00 | 41.80 | 15.00 | 627.00 |
B9 | 215.00 | 43.00 | 15.00 | 645.00 |
B10 | 193.00 | 38.60 | 15.00 | 579.00 |
B11 | 168.00 | 33.60 | 15.00 | 504.00 |
B12 | 240.00 | 48.00 | 15.00 | 720.00 |
C1 | 169.00 | 33.80 | 15.00 | 507.00 |
C2 | 230.00 | 46.00 | 15.00 | 690.00 |
C3 | 248.00 | 49.60 | 15.00 | 744.00 |
C4 | 204.00 | 40.80 | 15.00 | 612.00 |
C5 | 208.00 | 41.60 | 15.00 | 624.00 |
C6 | 139.00 | 27.80 | 15.00 | 417.00 |
C7 | 127.00 | 25.40 | 15.00 | 381.00 |
C8 | 174.00 | 34.80 | 15.00 | 522.00 |
C9 | 215.00 | 43.00 | 15.00 | 645.00 |
C10 | 202.00 | 40.40 | 15.00 | 606.00 |
C11 | 200.00 | 40.00 | 15.00 | 600.00 |
C12 | 179.00 | 35.80 | 15.00 | 537.00 |
D1 | 231.00 | 46.20 | 15.00 | 693.00 |
D2 | 234.00 | 46.80 | 15.00 | 702.00 |
D3 | 213.00 | 42.60 | 15.00 | 639.00 |
N37-1 | 201.00 | 40.20 | 15.00 | 603.00 |
N37-2 | 135.00 | 27.00 | 15.00 | 405.00 |
N37-3 | 194.00 | 38.80 | 15.00 | 582.00 |
N37-4 | 195.00 | 39.00 | 15.00 | 585.00 |
N37-5 | 165.00 | 33.00 | 15.00 | 495.00 |
N37-6 | 282.00 | 56.40 | 15.00 | 846.00 |
N37-7 | 206.00 | 41.20 | 15.00 | 618.00 |
N37-8 | 187.00 | 37.40 | 15.00 | 561.00 |
N37-9 | 256.00 | 51.20 | 15.00 | 768.00 |
N37-10 | 168.00 | 33.60 | 15.00 | 504.00 |
Capture set up[edit]
Probe calculation[edit]
Subset ID | Adjusted Multiplier based on qPCR (Multiplier E) | Actual amount of probe required (probe:target ratio, 100:1) | Multiplier E x amount probe required | Probe concentration (ng/ul) | Volume (ul) | Volume for 55rxn (ul) |
DMR.s1 | 0.05 | 2.23 | 0.11 | 1.86 | 0.06 | 3.30 |
DMR.s2 | 0.07 | 2.23 | 0.16 | 1.95 | 0.08 | 4.57 |
DMR.s3 | 0.23 | 18.90 | 4.41 | 23.20 | 0.19 | 10.44 |
DMR.s4 | 1.00 | 18.90 | 18.90 | 23.20 | 0.81 | 44.81 |
DMR.s5 | 4.83 | 18.90 | 91.29 | 29.20 | 3.13 | 171.94 |
DMR.Exp1 | 0.23 | 18.90 | 4.44 | 31.88 | 0.14 | 7.65 |
DMR.Exp2 | 0.19 | 18.90 | 3.50 | 24.11 | 0.15 | 8.00 |
DMR.Exp3 | 0.27 | 18.90 | 5.03 | 28.90 | 0.17 | 9.56 |
105nM Oligo suppressor | 1.00 | 55.00 | ||||
Total volume | 5.73 | 315.28 |
Reaction mix[edit]
Components | 1rxn (ul) | 55rxn mix (ul) |
10X AmpLigase Buffer | 2.50 | 137.50 |
DMR330k probe mix (ul) | 4.73 | 260.15 |
105nM Oligo suppressor | 1.00 | 55.00 |
Total volume (ul) | 8.23 | 452.65 |
sample ID | Final conc. (ng/ul) | Volume for 400ng(ul) | H2O (ul) | Total amount of bis-cvt gDNA (ng) | Probe mix (ul) | 10x Amligase buffer (ul) | 105nM Oligo suppressor (ul) | Total volume (ul) |
A1 | 36.80 | 10.87 | 5.90 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A2 | 63.20 | 6.33 | 10.44 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A3 | 41.60 | 9.62 | 7.15 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A4 | 44.80 | 8.93 | 7.84 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A5 | 35.20 | 11.36 | 5.41 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A6 | 43.60 | 9.17 | 7.60 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A7 | 48.00 | 8.33 | 8.44 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A8 | 44.00 | 9.09 | 7.68 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A9 | 73.00 | 5.48 | 11.29 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A10 | 44.00 | 9.09 | 7.68 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A11 | 43.20 | 9.26 | 7.51 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
A12 | 57.00 | 7.02 | 9.75 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B1 | 38.20 | 10.47 | 6.30 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B2 | 52.60 | 7.60 | 9.17 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B3 | 39.60 | 10.10 | 6.67 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B4 | 30.60 | 13.07 | 3.70 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B5 | 46.80 | 8.55 | 8.22 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B6 | 60.20 | 6.64 | 10.13 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B7 | 29.60 | 13.51 | 3.26 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B8 | 41.80 | 9.57 | 7.20 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B9 | 43.00 | 9.30 | 7.47 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B10 | 38.60 | 10.36 | 6.41 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B11 | 33.60 | 11.90 | 4.87 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
B12 | 48.00 | 8.33 | 8.44 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C1 | 33.80 | 11.83 | 4.94 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C2 | 46.00 | 8.70 | 8.07 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C3 | 49.60 | 8.06 | 8.71 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C4 | 40.80 | 9.80 | 6.97 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C5 | 41.60 | 9.62 | 7.15 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C6 | 27.80 | 14.39 | 2.38 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C7 | 25.40 | 15.75 | 1.02 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C8 | 34.80 | 11.49 | 5.28 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C9 | 43.00 | 9.30 | 7.47 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C10 | 40.40 | 9.90 | 6.87 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C11 | 40.00 | 10.00 | 6.77 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
C12 | 35.80 | 11.17 | 5.60 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
D1 | 46.20 | 8.66 | 8.11 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
D2 | 46.80 | 8.55 | 8.22 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
D3 | 42.60 | 9.39 | 7.38 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-1 | 40.20 | 9.95 | 6.82 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-2 | 27.00 | 14.81 | 1.96 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-3 | 38.80 | 10.31 | 6.46 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-4 | 39.00 | 10.26 | 6.51 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-5 | 33.00 | 12.12 | 4.65 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-6 | 56.40 | 7.09 | 9.68 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-7 | 41.20 | 9.71 | 7.06 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-8 | 37.40 | 10.70 | 6.07 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-9 | 51.20 | 7.81 | 8.96 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
N37-10 | 33.60 | 11.90 | 4.87 | 400.00 | 4.73 | 2.50 | 1.00 | 25.00 |
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_06_02) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_06_03)
SLN mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 150 |
AmpliTaq Stoffel | 10.00 | U/ul | 2.00 | U/ul | 30.00 |
AmpLigase | 5.00 | U/ul | 0.50 | U/ul | 15.00 |
dNTP | 1.00 | mM | 100.00 | uM | 15.00 |
10x AmpLigase Buffer | 10.00 | x | 1.00 | x | 15.00 |
H2O | 75.00 | ||||
Total | 150.00 |
- aliquot 15ul of SLN to strip tube, and add 2.5 ul to each tube.
- Note:
- There was something wrong with sample in well A9-A12. The volume of reaction in these four well was lower than 10ul, but before capture the levels of the reaction in every well were the same. I think that it's possible that the 96-well plate was not sealed very well especially the border of the plate, so the evaporation possibly occurred. There was proof that the reagents were not missing added to these four wells since one of them could be amplified, but not in a good yield. In the future, there should be more concerns about the plate sealing.
- It would be better if the samples will be rearranged from A1-H1, ..., A2-H2,.... rather than A1-A12, B1-B12. This because the multichannel pipette has 8 channels, so it would be easier to add samples in the same column at once. In addition, it's less possibility to miss adding reagents in well No.9-12
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX)[edit]
Components | 1rxn | 52 rxn mix |
Captured template | 8.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 104.00 |
10uM AmpR6.3 (IndX, X 1,2,3,…,49) | 2.00 | 0.00 |
50x SYBG | 0.80 | 41.60 |
2x Phusion MM | 50.00 | 2600.00 |
H2O | 37.20 | 1934.40 |
Total | 100.00 | Aliquot 90ul, add 8ul template and 2 ul of AmpR6.3 IndX |
Plate layout
A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | A11 | A12 |
AmpR6.3 Ind1 | AmpR6.3 Ind2 | AmpR6.3 Ind3 | AmpR6.3 Ind4 | AmpR6.3 Ind5 | AmpR6.3 Ind6 | AmpR6.3 Ind7 | AmpR6.3 Ind8 | AmpR6.3 Ind9 | AmpR6.3 Ind10 | AmpR6.3 Ind11 | AmpR6.3 Ind12 |
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
AmpR6.3 Ind13 | AmpR6.3 Ind14 | AmpR6.3 Ind15 | AmpR6.3 Ind16 | AmpR6.3 Ind17 | AmpR6.3 Ind18 | AmpR6.3 Ind19 | AmpR6.3 Ind20 | AmpR6.3 Ind21 | AmpR6.3 Ind22 | AmpR6.3 Ind23 | AmpR6.3 Ind24 |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
AmpR6.3 Ind25 | AmpR6.3 Ind26 | AmpR6.3 Ind27 | AmpR6.3 Ind28 | AmpR6.3 Ind29 | AmpR6.3 Ind30 | AmpR6.3 Ind31 | AmpR6.3 Ind32 | AmpR6.3 Ind33 | AmpR6.3 Ind34 | AmpR6.3 Ind35 | AmpR6.3 Ind36 |
D1 | D2 | D3 | NTC | ||||||||
AmpR6.3 Ind37 | AmpR6.3 Ind38 | AmpR6.3 Ind39 | AmpR6.3 Ind49 | ||||||||
N37-1 | N37-2 | N37-3 | N37-4 | N37-5 | N37-6 | N37-7 | N37-8 | N37-9 | N37-10 | ||
AmpR6.3 Ind40 | AmpR6.3 Ind41 | AmpR6.3 Ind42 | AmpR6.3 Ind43 | AmpR6.3 Ind44 | AmpR6.3 Ind45 | AmpR6.3 Ind46 | AmpR6.3 Ind47 | AmpR6.3 Ind48 | AmpR6.3 Ind49 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
Real-time PCR curves[edit]
File:A1-A8.png File:B1-B12.png File:C1-C12.png File:D1-D3.png File:N37-1-10 NTC.png
2011_06_06
- Amplify one more reaction for each sample excluded A9-A12 needed to be repeated for capturing
- Increase DNA captured template from 8ul to 10ul
Components | 1rxn | 47 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 94.00 |
10uM AmpR6.3 (IndX, X 1,2,3,…,49) | 2.00 | 0.00 |
50x SYBG | 0.80 | 37.60 |
2x Phusion MM | 50.00 | 2350.00 |
H2O | 35.20 | 1654.40 |
Total | 100.00 | Aliquot 88ul, add 10ul template and 2ul AmpR6.3IndX |
A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | ' | ' | ' | ' |
AmpR6.3 Ind1 | AmpR6.3 Ind2 | AmpR6.3 Ind3 | AmpR6.3 Ind4 | AmpR6.3 Ind5 | AmpR6.3 Ind6 | AmpR6.3 Ind7 | AmpR6.3 Ind8 | ||||
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
AmpR6.3 Ind13 | AmpR6.3 Ind14 | AmpR6.3 Ind15 | AmpR6.3 Ind16 | AmpR6.3 Ind17 | AmpR6.3 Ind18 | AmpR6.3 Ind19 | AmpR6.3 Ind20 | AmpR6.3 Ind21 | AmpR6.3 Ind22 | AmpR6.3 Ind23 | AmpR6.3 Ind24 |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
AmpR6.3 Ind25 | AmpR6.3 Ind26 | AmpR6.3 Ind27 | AmpR6.3 Ind28 | AmpR6.3 Ind29 | AmpR6.3 Ind30 | AmpR6.3 Ind31 | AmpR6.3 Ind32 | AmpR6.3 Ind33 | AmpR6.3 Ind34 | AmpR6.3 Ind35 | AmpR6.3 Ind36 |
D1 | D2 | D3 | |||||||||
AmpR6.3 Ind37 | AmpR6.3 Ind38 | AmpR6.3 Ind39 | |||||||||
N37-1 | N37-2 | N37-3 | N37-4 | N37-5 | N37-6 | N37-7 | N37-8 | N37-9 | N37-10 | ||
AmpR6.3 Ind40 | AmpR6.3 Ind41 | AmpR6.3 Ind42 | AmpR6.3 Ind43 | AmpR6.3 Ind44 | AmpR6.3 Ind45 | AmpR6.3 Ind46 | AmpR6.3 Ind47 | AmpR6.3 Ind48 | AmpR6.3 Ind49 |
File:PCR-round2 2011 06 06.png
Ct Values[edit]
- Calculated from first round and second round real-time PCR
Sample ID | Average Ct | Stdev |
A1 | 14.99 | NA |
A2 | 13.86 | 0.45 |
A3 | 15.01 | 0.09 |
A4 | 14.78 | 0.07 |
A5 | 13.42 | 0.78 |
A6 | 13.92 | 0.11 |
A7 | 14.58 | 0.04 |
A8 | 14.80 | 0.00 |
B1 | 13.17 | 0.52 |
B2 | 14.64 | 0.16 |
B3 | 15.23 | 0.11 |
B4 | 15.33 | 0.23 |
B5 | 15.37 | 0.21 |
B6 | 14.30 | 0.06 |
B7 | 15.81 | 0.11 |
B8 | 15.04 | 0.34 |
B9 | 15.31 | 0.44 |
B10 | 15.65 | 0.33 |
B11 | 16.04 | 0.42 |
B12 | 13.99 | 0.20 |
C1 | 12.81 | 0.37 |
C2 | 14.20 | 0.07 |
C3 | 14.62 | 0.08 |
C4 | 13.00 | 0.30 |
C5 | 15.46 | 0.11 |
C6 | 16.69 | 0.11 |
C7 | 16.89 | 0.32 |
C8 | 14.84 | 0.13 |
C9 | 15.66 | 0.33 |
C10 | 15.70 | 0.06 |
C11 | 16.05 | 0.30 |
C12 | 16.08 | 0.02 |
D1 | 15.47 | 0.57 |
D2 | 15.66 | 0.25 |
D3 | 15.53 | 0.45 |
N37-1 | 14.46 | 0.06 |
N37-2 | 15.26 | 0.23 |
N37-3 | 13.36 | 0.51 |
N37-4 | 14.89 | 0.02 |
N37-5 | 14.21 | 0.02 |
N37-6 | 15.74 | 0.20 |
N37-7 | 14.99 | 0.13 |
N37-8 | 15.60 | 0.08 |
N37-9 | 15.99 | 0.65 |
N37-10 | 15.40 | 1.06 |
- Continued on 2011_06_06: [[1]]