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==== Sample list ==== * WT = wild type * T= TKO * Number after WT or T like W10, T100 is numb rod nuclei the rest are W or T with single nucleus and running order number. :{| {{table}} border = 1 | WT_1||WT_2||WT_3||WT_4||WT_5||WT_6||WT_7||WT_8 |- | WT_9||WT_10||WT_11||WT_12||T_1||T_2||T_3||T_4 |- | T_5||T_6||WT10_1||WT10_2||WT100||0nu||NTC|| |- | T_7||T_8||T_9||T_10||T_11||T_12||T10||T100 |- | NTC||0nu|||||||||||| |} * I handled sample WT-1 --> NTC (23 samples) * Youjin handled sample T7 --> 0nu (10 samples) * I prepared the same master mix then aliquot for Youjin for every step. * Note that Youjin did not pick 0 nuclei from the nucleus suspension which is ideal negative control to make sure that the signals do not come from buffer or other sources. We therefore use lysis buffer that I aliquot as bedding buffer as 0nu control. * For NTC, I aliquot lysis buffer from original tube of lysis buffer and add H2O instead of pick nuclei. :- Add 1.05 ul of protease and unmeth lambda DNA mix to each tube. No need to pipette! :- Spin down the tube on PCR rack at 2,000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min ::- I tried to avoid pipetting up and down to mix the reaction to prevent nuclei/DNA lost. ::- I spin down the plate before and after mixing quite long to make sure that all reagents were collected to the bottom of the tube :- ''Incubate at 50C for 3hr''<br> :- ''Heat inactivate at 75C for 30min'' :- Set program to hold at 15C :- Spin down the plate at 2,000rpm for 1min before continuing to next step * Note that before cell lysis and after cell lysis MspI digestion, I need to spin the tube containing single nucleus at high speed and long time to make sure that I do not lose nucleus. <br>
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