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= 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 Link to calendar]] * Continued from: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-19]] :[[File:MONOD-sensitivity-test-plans.png| 700px]] : - 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 === {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''500:1''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''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 ==== :{| {{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 two thermocyclers, standard capture protocol and multiple rounds of circularization protocol. ---- ==== 2) No amplification before BSPP capture ==== :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''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 ===== :{| {{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 (ul)''' | align="center" style="background:#f0f0f0;"|'''MONOD_GP1_V4<br>probe (19.60ng)''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase<br>Buffer''' | align="center" style="background:#f0f0f0;"|'''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 ===== :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''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 ===== '''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 ===== '''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) == * I will only test 100ng, 200ng & 300ng templates * Prepare PCR mix with MONOD_V3_12k experiment {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''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<br> '''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
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