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==Reagent Preparation== dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x30 15ul 3ul 3ul 6ul 3ul ==Overview== *Bisulfite Conversion of DNA *Capturing Reaction ===Calculation=== Probe:target ratio 200:1 (according to Alice, 100:1 works too) *Billy uses 250ng NIH_3T3_gDNA Template(100ng/uL) Use 200ng total, so = 2uL *One genome mass = 3pg *First calculate the # of genome your target represents *For 200ng, it represents 200ng/3pg=66666.67 genomes *You want to have 200x the probes as the target 200ng/3pg*200 *You want every single probe to target ratio to be 200:1 200ng/3pg*200*55,000 Ms_Meth 22ng/uL 55,000Probes 100nt Since 1 base = 330Da (for single stranded DNA, 660Da for ds), the concentration of probe is 22 ng/ul /(100x330) = 0.000666667 = 666.67nM # of molecules per mole = 6X10^23 # of probes needed/ concentration of probes = Volume of probes needed the volume the probe needed is 200ng/3pg x 200 x 55,000 / [(6x10^23)x (666.67*10^-9M)]*10^6 =1.83uL ==Procedures== ==Bilsulfite Conversion with EZ DNA Methylation-Gold Kit== *Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent vortex for 10M *Add 24mL of 100% ethanol to the 6mL M-Wash Buffer concentrate *Add 130ul of CT conversion reagent to samples. If your DNA sample is less than 20uL make up the difference with H2O *Mix the sample and perform *98C 10min -> 64C 150min -> 4c overnight. *Add 600ul M-Binding buffer to spin columns. *Add the converted samples to the columns, close the gap and mix by inverting several times. *Spin at 15,000rpm for 30sec, discard flow through *Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through. *Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec. *Add 200ul M-Wash Buffer, spin for 30 sec. *Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec. *Measure the DNA with Nanodrop: RNA ==Capturing System Setup== CT Converted Ms-gDNA 10X AmpLigase buffer Ms_Meth Probe (242.4nM) H2O Total 95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> <BR>4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min -> <BR>add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold
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