Noi/NOTES/2014-3-29

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Revision as of 17:53, 31 March 2014 by >Noi (→‎Probe calculation)
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DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)

[Link to calendar]

  • Continued from: [[1]]
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

  1. Low-magnitude amplification before BSPP capture
  2. 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 on two thermocyclers, standard capture protocol and multiple rounds of circularization protocol.