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==Procedures== * Diltue Jurkat gDNA to equal amount of gDNA from 100 cells 100cells equivalent to 600pg or 0.6ng gDNA ---- '''10ng/uL''' Dilute 10uL stock gDNA(100ng/uL) with 90uL RNAse free H2O ---- '''1ng/uL''' Dilute 10uL stock gDNA(10ng/uL) with 90uL RNAse free H2O ---- '''0.1ng/uL''' Dilute 10uL stock gDNA(1ng/uL) with 90uL RNAse free H2O ---- *Bisulfite Conversion of DNA PCR Tube A - 200ng Jurkat gDNA 2uL 100ng/uL gDNA 18uL H20 130uL CT Conversion Reagent PCR Tube B 0.6ng Jurkat gDNA 6uL 0.1ng/uL gDNA 14uL H2O 130uL CT converison Reagent Vortex and spin down Perform reaction in thermocycler *Run>Alan>METH Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr Add 700uL of M-Bindin Buffer into a IC Column (Increase from 600uL, should increase yield) Load 150uL of samples into IC column '''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES''' Centrifuge at 20,000 rcf 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 Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge CPG30K Dilution * Diltue CPG30K ---- '''0.7ng/uL''' Dilute 10uL stock gDNA(7ng/uL) with 90uL RNAse free H2O ---- '''0.07ng/uL''' Dilute 10uL stock gDNA(0.7ng/uL) with 90uL RNAse free H2O *Setup the Capture Reaction {| border="1" align="center" |rowspan="6" align="center"|Tube A | align="center" style="background:#f0f0f0;"|'''Content ''' | align="center" style="background:#f0f0f0;"|'''Volume ''' |- |Template||align="right"|10uL |- |10X Ligase Buffer||align="right"|1uL |- |7ng/uL CPG30K||align="right"|7.24uL |- |RNAse free H2O||align="right"|1.76uL |- |Mineral Oil||align="right"|20uL |- |Total Volume | colspan="2" align="right"| 40uL |} {| border="1" align="center" |rowspan="6" align="center"|Tube B | align="center" style="background:#f0f0f0;"|'''Content ''' | align="center" style="background:#f0f0f0;"|'''Volume ''' |- ||Template||align="right"|10uL |- ||10X Ligase Buffer||align="right"|1uL |- ||0.07ng/uL CPG30K||align="right"|4.29uL |- ||RNAse free H2O||align="right"|4.71uL |- ||Mineral Oil||align="right"|20uL |- |Total Volume | colspan="2" align="right"| 40uL |} *Thermocycler *Run>Alan>CAP Step1 95C, 10m Step2 55C, hold at least 16 hrs *Add 1uL SLN mix Step3 55C, 4hr Step4 95C, 1m Step5 55C, 4hr Step6 Go back to step 4 repeat 8 times Step7 95C, 1m Step8 37C, 1m *Add 2uL Exonuclease I/III mix Step9 37C, 2hr Step10 94C, 5m Step11 4C, hold *PCR A+B Template 10ul 2X iProof Mastermix 50ul AmpF6.2SoL (10uM) 4ul AmpR6.2SoL (10uM) 4ul 50X SYBG I 0.8ul H2O 31.2ul Step1 98C, 30s Step2 98C, 10s Step3 58C, 20s Step4 72C, 20s Step5 Go back to step 2 repeat 7 times Step6 98C, 10s Step7 72C, 20s Step8 Go back to step 6 repeat 9 times Step9 72C, 5m Step10 15C, 0m *Gel Electrophoresis Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank Ladder Sample 6X Loading Dye ----------------------------------------------------------------- Total
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