Revision as of 17:54, 31 March 2014 by >Noi
DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)
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- File:MONOD-sensitivity-test-plans.png
- - For all bis-DNA input, I will use the same concentration of probe in capture reaction by using 500:1 probe:target ratio for 300ng bis-DNA
BSPP capture set up
Probe calculation
Probe:target
|
500:1
|
Unit
|
Calculation
|
Probe size (number of individual probe in oligo mix) |
7,110 |
probes |
|
Human bis-cvt gDNA required |
300 |
ng |
|
Human gDNA MW |
1.822E+12 |
g/mole |
3.0E+09 * 607.4Da/bp
|
Human gDNA( 300ng) |
1.646E-19 |
mole |
300E-09g / 1.822E+12g/mole
|
Probe required (500:1) |
8.232E-17 |
mole |
500 * 1.098E-19mole
|
Probe mix MW (size 7,110probes, 110nt) |
2.381E+08 |
g/mole |
7,110 probes * (110bp *303.7Da/bp+79Da)
|
Mass of probe required |
1.960E-08 |
g |
8.232E-17mole * 2.381E+08g/mole
|
Mass of probe required |
19.60 |
ng |
|
Probe conc |
6.87 |
ng/ul |
|
Volume of probe |
2.85 |
ul |
|
Two parts of experiment
- Low-magnitude amplification before BSPP capture
- No amplification before capture
1) Low-magnitude amplification before BSPP capture
Sampe description
|
Tube IDs
|
5ng_STD BSPP capture |
5a-S
|
10ng_STD BSPP capture |
10a-S
|
25ng_STD BSPP capture |
25a-S
|
50ng_STD BSPP capture |
50a-S
|
5ng_modified BSPP capture |
5a-M
|
10ng_modified BSPP capture |
10a-M
|
25ng_modified BSPP capture |
25a-M
|
50ng_modified BSPP capture |
50a-M
|
NTC |
NTCa-S
|
- Dilute bis-JURKAT 38.3ng/ul --> 2.5, 5.0, 25 ng/ul
Stock 38.3ng/ul
|
H2O
|
Total volume (ul)
|
Final conc. (ng/ul)
|
6.53 |
93.47 |
100.00 |
2.50
|
13.05 |
86.95 |
100.00 |
5.00
|
32.64 |
17.36 |
50.00 |
25.00
|
- Mix bis-JURKAT with Ampligase buffer and 2uM N9 random primers
bis-JURKAT
|
Conc. (ng/ul)
|
Amount required (ng)
|
Volume forXXng (ul)
|
bis-JURKAT
|
H2O
|
5a-S |
2.50 |
5.00 |
2.00 |
5a-S |
4.30
|
10a-S |
5.00 |
10.00 |
2.00 |
10a-S |
4.30
|
25a-S |
5.00 |
25.00 |
5.00 |
25a-S |
1.30
|
50a-S |
25.00 |
50.00 |
2.00 |
50a-S |
4.30
|
5a-M |
2.50 |
5.00 |
2.00 |
5a-M |
4.30
|
10a-M |
5.00 |
10.00 |
2.00 |
10a-M |
4.30
|
25a-M |
5.00 |
25.00 |
5.00 |
25a-M |
1.30
|
50a-M |
25.00 |
50.00 |
2.00 |
50a-M |
4.30
|
NTCa-S |
0.00 |
0.00 |
0.00 |
NTCa-S |
6.30
|
Mix I: Ampligase buffer & N9 primer mix
N9 & Buffer Mix
|
1x rxn
|
12x rxn
|
10X AmpLigase Buffer |
1.00 |
12.00
|
100uM N9 |
0.20 |
2.40
|
Total |
1.20 |
|
Mix II: Klenow, exo- & dNTP mix
Klenow & dNTP mix
|
1x rxn
|
12x rxn
|
10mM dNTP Mix |
0.20 |
2.40
|
Klenow Fragment, exo - (5U/ul) |
0.50 |
6.00
|
H2O |
1.80 |
21.60
|
Total |
2.50 |
|
Mix III: MONOD_GP1_V4 BSPP & Ampligase buffer mix
Probe & Buffer Mix
|
1x rxn
|
9.5x rxn
|
10X AmpLigase Buffer |
1.00 |
9.50
|
MONOD_GP1_V4 probe (19.60ng) |
2.85 |
27.08
|
H2O |
6.15 |
58.43
|
Total |
10.00 |
|
- - Add 1.2ul of Ampligase/N9 primer mix to bis-JURKAT
- - 94C, 3 min --> 25C 3min
- - Add 2.5ul of Klenow Fragment & dNTP mix (I moved the tube from thermocycler and mix samples at RT)
- - 25C, 1h --> 75C, 20min
- - Add 10ul of probe & buffer mix
- - Layer capture tubes with 2 drops of mineral oil
- - Place the tube on two thermocyclers, standard capture protocol and multiple rounds of circularization protocol.
2) No amplification before BSPP capture