Noi/NOTES/2014-3-29: Difference between revisions
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>Noi (Created page with "= DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture) = [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Li...") |
>Noi |
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| Volume of probe||2.85||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 ==== | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sampe description''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Stock 38.3ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''bis-JURKAT''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount required (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume forXXng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''bis-JURKAT''' | |||
| align="center" style="background:#f0f0f0;"|'''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 ===== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''N9 & Buffer Mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''12x rxn''' | |||
|- | |||
| 10X AmpLigase Buffer||1.00||12.00 | |||
|- | |||
| 100uM N9||0.20||2.40 | |||
|- | |||
| Total||1.20|| | |||
|} | |||
===== Mix II: Klenow, exo- & dNTP mix ===== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Klenow & dNTP mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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 ===== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe & Buffer Mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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. |
Revision as of 17:53, 31 March 2014
DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)
- 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
- 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 on two thermocyclers, standard capture protocol and multiple rounds of circularization protocol.