Noi/NOTES/2011-5-28
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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
- 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
- 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
Probe calculation
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
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; 50uM 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
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.