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==Procedures== *Turn on the incubator heat it up to 50C *Cell Preparation Resuspended cells in T25 flask by repeat pipetting Perform cell counting *Cell Count of GM20431 P20: 207,656 cells/mL To get 1,000,000 cells: 1,000,000/(207,656cells/mL)=4.816mL aspirate 4mL + 816uL cell suspension and transfer to a 50mL centrifuge tube Spin down at 10,000 rpm for 5 mins Aspirate supernatant completely Resuspended cells with 1000uL UV treated PBS (1000cells/uL) *Cell Dilution Performed cell dilution to reach concentration of 11.11 cells/uL and 1.11 cells/uL *111.11 cells/uL Dilute 10uL (1000cells/uL)cell suspension with 80uL UV TREATED PBS IN PCR TUBE *11.11 cells/uL Dilute 10uL (111.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE *1.11 cells/uL Dilute 10uL (11.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE *Sample Digestion with Proteinase K 1rxn x 6 -------------------------------------------- RNAse-free H2O 0.0 0.0 Protinase K 1.0 1.0 M-Digestion Buffer (2X) 10.0 10.0 1.11 cells/uL 9.0 -------------------------------------------- 20uL 20uL Vortex and spindown Incubate the samples at 50C for 20 mins *Bisulfite Conversion of DNA Add in 130uL of CT conversion Reagent Solution directly to the digested samples Vortex and spin down Perform reaction in thermocycler Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr Add 600uL of M-Bindin Buffer into a IC Column Load 150uL of samples into IC column '''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES''' Centrifuge at 20,000g for 30s Discard flow through Add 100uL M-Wash Buffer to column Repeat Centrifuge Add 200uL of M-Desulphonation Buffer to column let stand at RT for 20m Repeat centrifuge step Add 200uL of M-Wash Buffer to the column Repeat Centrifuge [REPEAT WASHING STIP FOR ONE SET ONLY] Place column in a 1.5mL tube Add in 8uL of M-Elution Buffer directly to the column matix Repeat Centrifuge *PCR Sample A 10 cell GM20431 (Single Washing) A B C ------------------------------------------------- CHR22 CHR21 CHR8 2X iQ Super Mix 20 20 20 Primer F (3.3uM) 6 6 6 Primer R (3.3uM) 6 6 6 gDNA 8 8 8 ------------------------------------------------- Total 40uL Sample B 10 cell GM20431 (Double Washing) A B C ------------------------------------------------- CHR22 CHR21 CHR8 2X iQ Super Mix 20 20 20 Primer F (3.3uM) 6 6 6 Primer R (3.3uM) 6 6 6 gDNA 8 8 8 ------------------------------------------------- Total 40uL A-CHR22 F/R B-CHR21 F/R C-CHR8 F/R Perform PCR reaction in thermocycler Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever *Gel Electrophoresis Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank AA AB AC BA BB BC Ladder Sample 0 10 10 10 10 10 10 3 6X Loading Dye 0 2 2 2 2 2 2 3 ----------------------------------------------------------------- Total 14uL 9uL *Run gel at 135 V for 20 min.
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