Noi/NOTES/2014-3-29
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DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)[edit]
- 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]
- Low-magnitude amplification before BSPP capture
- 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
BufferTotal (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