Revision as of 16:07, 27 August 2014 by >Noi
BSPP LMS cluster capture of colon cancer primary tumor DNA[edit]
Calendar
Probe prep
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
|