Noi/NOTES/2014-3-29

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DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)[edit]

[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[edit]

Probe calculation[edit]

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[edit]

  1. Low-magnitude amplification before BSPP capture
  2. No amplification before capture

1) Low-magnitude amplification before BSPP capture[edit]

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[edit]
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[edit]
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[edit]
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[edit]

Samples Code
5ng_STD BSPP capture 5-S
10ng_STD BSPP capture 10-S
25ng_STD BSPP capture 25-S
50ng_STD BSPP capture 50-S
100ng_STD BSPP capture 100-S
200ng_STD BSPP capture 200-S
300ng_STD BSPP capture 300-S
5ng_modified BSPP capture 5-M
10ng_modified BSPP capture 10-M
25ng_modified BSPP capture 25-M
50ng_modified BSPP capture 50-M
NTC NTC-S
Capture set up[edit]
bis-JURKAT Conc. (ng/ul) Amount required (ng) Volume forXXng (ul) bis-JURKAT H2O (ul) MONOD_GP1_V4
probe (19.60ng)
10X AmpLigase
Buffer
Total (ul)
5-S 2.50 5.00 2.00 5-S 13.15 2.85 2.00 20.00
10-S 5.00 10.00 2.00 10-S 13.15 2.85 2.00 20.00
25-S 5.00 25.00 5.00 25-S 10.15 2.85 2.00 20.00
50-S 25.00 50.00 2.00 50-S 13.15 2.85 2.00 20.00
100-S 38.30 100.00 2.61 100-S 12.54 2.85 2.00 20.00
200-S 38.30 200.00 5.22 200-S 9.93 2.85 2.00 20.00
300-S 38.30 300.00 7.83 300-S 7.32 2.85 2.00 20.00
5-M 2.50 5.00 2.00 5-M 13.15 2.85 2.00 20.00
10-M 5.00 10.00 2.00 10-M 13.15 2.85 2.00 20.00
25-M 5.00 25.00 5.00 25-M 10.15 2.85 2.00 20.00
50-M 25.00 50.00 2.00 50-M 13.15 2.85 2.00 20.00
NTC-S 0.00 0.00 0.00 NTC-S 15.15 2.85 2.00 20.00
Probe & AmpLigase Buffer mix[edit]
Components Volume (ul) 12.5x
10X AmpLigase Buffer 2.00 25.00
MONOD_GP1-V4 (6.87 ng/ul) 2.85 35.63
Total 4.85
Split samples into two groups, incubate on thermocycler following standard BSPP capture (s) and modified BSPP capture (multiple round of circularization, m)
Standard BSPP capture[edit]
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
Modified BSPP capture[edit]
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 4h
-> 4x of [95C, 1min -> 55C, 4h)
-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

Amplification (TEST)[edit]

  • I will only test 100ng, 200ng & 300ng templates
  • Prepare PCR mix with MONOD_V3_12k experiment
Components 1x rxn 19x rxn mix
Captured template 4.00 0.00
10uM AmpF6.4Sol 0.50 9.50
10uM AmpR6.3Indx1 0.50 9.50
2X KAPA SYBR MM 12.50 237.50
H2O 7.50 142.50
Total 25.00

- Aliquot 21ul, add 4ul of circularized DNA

Program (Eppendorf Realplex)

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
  • This experiment failed because I put the tubes on the wrong slots, could not read signal and overamplified (upto 30 cycles)
  • I have repeated experiment on April 10, 2014