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==Procedures== *Cell Preparation Resuspended cells in T25 flask by repeat pipetting Perform cell counting *Cell Countof GM20431 P15: 346,430 cells/mL To get 1,000,000 cells: 1,000,000/(346,430/mL)=2.886mL aspirate 2mL + 886uL cell suspension and transfer to a 50mL centrifuge tube Spin down at 10,000rpm for 3 mins Aspirate supernatant completely Resuspended cells with 1000uL UV treated PBS (1000cells/uL) *Cell Dilution Performed cell dilution to reach concentration of 111.11 cells/uL Dilute 8uL (1000cells/uL)cell suspension with 800uL UV treated PBS *Sample Digestion with Proteinase K 1rxn x 2 ------------------------------------ RNAse-free H2O 0.0 Protinase K 1.0 M-Digestion Buffer (2X) 10.0 ------------------------------------ Mix by repeat pipetting Cells 9.0 ------------------------------------ 20uL Incubate the samples at 50C for 20 mins *Bisulfite Conversion of DNA Add in 130uL of CT conversion Reagent Solution into a labeled PCR tube Extract 20uL of sample supernatant Mix by repeat pipetting Centrifuge briefly to ensure no droplets are in the cap or side of the tube Perform reaction in thermocycler Step1 98C, 8m Step2 64C, 3.5hr Step3 62C, 1m Step4 4C, storage for up to 20 hr Add 600uL of M-Bindin Buffer into a IC Column and place column into a collection tube Load samples into IC column Close cap and mix by inverting the column several times Centrifuge at (max speed) 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 step Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge *PCR Sample A 1,000cell GM20431 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL Sample B 1,000cell GM20431 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL Sample C 6ng Jurkat gDNA 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL Sample D 6ng Jurkat gDNA 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- 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 *1st and 2nd Gel Electrophoresis Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank AA AB AC CA CB CC Ladder Sample 0 6 6 6 6 6 6 3 0.5X TBE Buffer 0 4 4 4 4 4 4 3 6X Loading Dye 0 4 4 4 4 4 4 3 ----------------------------------------------------------------- Total 14uL 9uL Gel 2 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank BA BB BC DA DB DC Ladder Sample 0 6 6 6 6 6 6 3 0.5X TBE Buffer 0 4 4 4 4 4 4 3 6X Loading Dye 0 4 4 4 4 4 4 3 ----------------------------------------------------------------- Total 14uL 9uL **Run both gels at the same time at 135 V for 20 min.
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