Revision as of 16:18, 3 March 2014 by >Noi
DNA methylation assay - MONOD Project (BSPP capture)
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Sample list
- - Cancer cell lines (5 samples)
- - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- - Blood samples (3 samples)
Bisulfite conversion
- I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit
Bisulfite conversion set up
Samples
|
Conc. (ng/ul)
|
Volume for 600ng (ul)
|
Go to tube
|
H2O
|
Lightning Conv Reagent
|
CANC_1 |
47.70 |
12.58 |
MN#1 |
7.42 |
130.00
|
CANC_2 |
45.40 |
13.22 |
MN#2 |
6.78 |
130.00
|
CANC_3 |
41.40 |
14.49 |
MN#3 |
5.51 |
130.00
|
CANC_4 |
47.10 |
12.74 |
MN#4 |
7.26 |
130.00
|
CANC_5 |
68.20 |
8.80 |
MN#5 |
11.20 |
130.00
|
SR-3 |
62.20 |
9.65 |
MN#6 |
10.35 |
130.00
|
MA-16 |
85.00 |
7.06 |
MN#7 |
12.94 |
130.00
|
MA-17 |
49.40 |
12.15 |
MN#8 |
7.85 |
130.00
|
F10 |
106.00 |
5.66 |
MN#9 |
14.34 |
130.00
|
G10 |
108.00 |
5.56 |
MN#10 |
14.44 |
130.00
|
G11 |
82.60 |
7.26 |
MN#11 |
12.74 |
130.00
|
NTC |
0 |
0 |
MN#12 |
20.00 |
130.00
|
- I elute with Elution buffer 16ul
- I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.
Qubit ss DNA assay
- Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
Sample
|
Conc. in the Qubit
|
'
|
uL used
|
Dilution
|
Sample conc.
|
'
|
Yields in 13ul (ng)
|
% recovery
|
MN_1 |
175 |
ng/mL |
1 |
200 |
34.9 |
ng/ul |
453.7 |
75.62
|
MN_2 |
122 |
ng/mL |
1 |
200 |
24.5 |
ug/mL |
318.5 |
53.08
|
MN_3 |
122 |
ng/mL |
1 |
200 |
24.5 |
ug/mL |
318.5 |
53.08
|
MN_4 |
87.6 |
ng/mL |
1 |
200 |
17.5 |
ug/mL |
227.5 |
37.92
|
MN_5 |
108 |
ng/mL |
1 |
200 |
21.7 |
ug/mL |
282.1 |
47.02
|
MN_6 |
131 |
ng/mL |
1 |
200 |
26.2 |
ug/mL |
340.6 |
56.77
|
MN_7 |
107 |
ng/mL |
1 |
200 |
21.4 |
ug/mL |
278.2 |
46.37
|
MN_8 |
71.4 |
ng/mL |
1 |
200 |
14.3 |
ug/mL |
185.9 |
30.98
|
MN_9 |
61.5 |
ng/mL |
1 |
200 |
12.3 |
ug/mL |
159.9 |
26.65
|
MN_10 |
173 |
ng/mL |
1 |
200 |
34.6 |
ug/mL |
449.8 |
74.97
|
MN_11 |
72.9 |
ng/mL |
1 |
200 |
14.6 |
ug/mL |
189.8 |
31.63
|
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
BSPP capture set up
Probe calculation
Probe:taeget
|
200:1
|
Unit
|
Calculation
|
Probe size (number of individual probe in oligo mix) |
83447 |
probes |
|
Human bis-cvt gDNA required |
200 |
ng |
|
Human gDNA MW |
1.822E+12 |
g/mole |
3.0E+09 * 607.4Da/bp
|
Human gDNA( 200ng) |
1.098E-19 |
mole |
200E-09g / 1.822E+12g/mole
|
Probe required (200:1) |
2.195E-17 |
mole |
200 * 1.098E-19mole
|
Probe mix MW (size 83447probes, 110nt) |
2.788E+09 |
g/mole |
83447 probes * 110bp *303.7Da/bp
|
Mass of probe required |
6.119E-08 |
g |
2.195E-17mole * 2.788E+09g/mole
|
Mass of probe required |
61.20 |
ng |
|
Probe conc |
11.9 |
ng/ul |
|
Volume of probe |
5.14 |
ul |
|
Probe & AmpLigase Buffer mix
Components
|
Volume (ul)
|
12.5x
|
10X AmpLigase Buffer |
2.00 |
25.00
|
MONOD (11.90ng/ul) |
5.14 |
64.25
|
Toal |
7.14 |
|
Bis-cvt samples
|
Conc. (ng/ul)
|
Volume for 200ng (ul)
|
Bis-cvt samples
|
H2O (ul)
|
MONOD probe (61.20ng)
|
10X AmpLigase Buffer
|
Total (ul)
|
Converted DNA amount (ng)
|
MN_1 |
34.90 |
5.73 |
MN_1 |
7.13 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_2 |
24.50 |
8.16 |
MN_2 |
4.70 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_3 |
24.50 |
8.16 |
MN_3 |
4.70 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_4 |
17.50 |
11.43 |
MN_4 |
1.43 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_5 |
21.70 |
9.22 |
MN_5 |
3.64 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_6 |
26.20 |
7.63 |
MN_6 |
5.23 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_7 |
21.40 |
9.35 |
MN_7 |
3.51 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_8 |
14.30 |
13.00 |
MN_8 |
0.00 |
5.14 |
2.00 |
20.14 |
185.90
|
MN_9 |
12.30 |
13.00 |
MN_9 |
0.00 |
5.14 |
2.00 |
20.14 |
159.90
|
MN_10 |
34.60 |
5.78 |
MN_10 |
7.08 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_11 |
14.60 |
13.00 |
MN_11 |
0.00 |
5.14 |
2.00 |
20.14 |
189.80
|
MN_12 |
0.00 |
0.00 |
MN_12 |
12.86 |
5.14 |
2.00 |
20.00 |
0.00
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 3ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- Note: Concentration of Hemo Klentaq was not provided, so I assumeed it has the same concentration as Stoffel Fragment