Noi/NOTES/2014-8-27
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BSPP LMS cluster capture of colon cancer primary tumor DNA[edit]
Experimental plan[edit]
- Use MONOD V5 set A to capture DNA from colon tumor DNA
- Test sensitivity of BSPP capture of this probe set on 10, 20, 100 and 200ng
Bisulfite conversion and ssDNA quantification[edit]
- I did bisulfite conversion of 5 colon cancer DNA sample in duplicates using EZ DNA Methylation-Lightning™ Kit
- I followed the instruction provided by manufacturer
Sample ID | Conc. (ng/ul) | Volume for 600ng (ul) | H2O to 20ul |
6-T-1 | 43.40 | 13.82 | 6.18 |
6-T-2 | 31.60 | 18.99 | 1.01 |
6-T-3 | 31.50 | 19.05 | 0.95 |
6-T-4 | 41.70 | 14.39 | 5.61 |
6-T-5 | 30.50 | 19.67 | 0.33 |
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. Continue to desulfonation
- - Elute with 18ul Elution Buffer
- - The volume left after Elution ~16ul
- - Quantify concentration and recovery yield by Qubit ssDNA assay kit
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Conc. (ng/ul) | Yield in 16ul (ng) | % Recovery |
6-T-1_1 | 136 | ng/mL | 1 | 200 | 27.20 | 435.20 | 72.53 |
6-T-1_2 | 144 | ng/mL | 1 | 200 | 28.90 | 462.40 | 77.07 |
6-T-2_1 | 92.2 | ng/mL | 1 | 200 | 18.40 | 294.40 | 49.07 |
6-T-2_2 | 95 | ng/mL | 1 | 200 | 19.00 | 304.00 | 50.67 |
6-T-3_1 | 137 | ng/mL | 1 | 200 | 27.40 | 438.40 | 73.07 |
6-T-3_2 | 131 | ng/mL | 1 | 200 | 26.30 | 420.80 | 70.13 |
6-T-4_1 | 117 | ng/mL | 1 | 200 | 23.40 | 374.40 | 62.40 |
6-T-4_2 | 134 | ng/mL | 1 | 200 | 26.80 | 428.80 | 71.47 |
6-T-5_1 | 133 | ng/mL | 1 | 200 | 26.60 | 425.60 | 70.93 |
6-T-5_2 | 140 | ng/mL | 1 | 200 | 27.90 | 446.40 | 74.40 |
BSPP capture set up[edit]
Probe calculation[edit]
Probe:target | 200 : 1 | Unit, calculation |
Probe size | 73,574 | |
Template | 10 | ng |
Human gDNA MW | 1.950E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (10ng) | 5.128E-21 | mol (10ng/1.950E+12 g/mol) |
Probe required (200:1) | 1.026E-18 | mol (2.5641E-20 * 200) |
Probe MW (73,574 probes, 110nt) | 2.636E+09 | g/mol (73,574 probes * (110nt * 325Da/nt + 79Da)) |
Amount probe required | 2.704E-09 | g |
Amount probe required | 2.7 | ng |
Input bis-DNA (ng) | MONOD V5 set A amount (ng) |
10 | 2.70 |
20 | 5.40 |
100 | 27.00 |
200 | 54.00 |