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AlanFung:LabNotes/Capturing/CPG97K Low-input/2009-10-24
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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 *CT converted Jurkat gDNA (200ng/ul) *CT converted GM20431 (11.6ng/ul) *According to Ida low-input works on 50ng, test with 100ng first *to get 100ng *Need- 0.5ul Jurkat gDNA * 8.62ul GM20431 gDNA *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 Qubit ssDNA kit: *'''CT Converted GM20431 gDNA concentration: 11.6ng/ul''' *'''Concentration of CT converted Jurkat gDNA: 200ng/ul''' *'''Concentration of N6 hexamer: 10ng/ul''' ==Capturing System Setup== *Prepare total volume up to 15ul {| {{table}} | ||50ng input||||||||30ng input|||| |- | Capturing System||Jurkat||GM20431||Negative Control||||Jurkat||GM20431||Negative Control |- | Volume of N6 (10ng/ul)||5.000||5.000||0.000||||7.000||7.000||0.000 |- | Volume of probes (0.7ng/ul)||3.929||3.929||3.929||||2.357||2.357||2.357 |- | Volume of templates||2.500||4.310||0.000||||1.500||2.586||0.000 |- | Volume of H20||2.071||0.261||9.571||||2.643||1.557||11.143 |- | Volume of 10X Ampligase buffer||1.500||1.500||1.500||||1.500||1.500||1.500 |- | Total Volume||15.000||15.000||15.000||||15.000||15.000||15.000 |- | |||||||||||||| |- | ||10ng input||||||||6ng input|||| |- | Capturing System||Jurkat||GM20431||Negative Control||||Jurkat||GM20431||Negative Control |- | Volume of N6 (10ng/ul)||9.000||9.000||0.000||||9.400||9.400||0.000 |- | Volume of probes||0.786||0.786||0.786||||0.471||0.471||0.471 |- | Volume of templates||0.500||0.862||0.000||||0.300||0.517||0.000 |- | Volume of H20||3.214||2.852||12.714||||3.329||3.111||13.029 |- | Volume of 10X Ampligase buffer||1.500||1.500||1.500||||1.500||1.500||1.500 |- | Total Volume||15.000||15.000||15.000||||15.000||15.000||15.000 |- | |} 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. ==PCR(01/26/2009)== *'''Scale down template to 2ul and run till saturation, to observe the result of different amount of template input''' {| {{table}} | ||1well (ul)||X15||/5 |- | Template||10||150||30 |- | 2X Phusion||50||750||150 |- | AmpF6.2SoL (10uM)||4||60||12 |- | AmpR6.2Sol (10uM)||4||60||12 |- | 50X SYBG I||0.8||12||2.4 |- | H2O||31.2||468||93.6 |- | |} *Plate Setup {| {{table}} | Setup|| |- | 50JT||10G |- | 50J||10N |- | 50G||6J |- | 50N||6G |- | 30J||6N |- | 30G|| |- | 30N|| |- | 10J|| |- | |} 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 20-> 72C 5 min -> 15C hold. *Update after 11/09/09 meeting (Need to move on to the next step on the middle panel) *New qPCR result [[Image:11_10_09_ALL.jpg]] ==11/6/09== ==11/7/09== *Repeat amplification using all of the 50ng captured product {| {{table}} | ||1well (ul)||X3||X1.1 |- | Template||10 |- | 2X Phusion||50||150||165 |- | AmpF6.2SoL (10uM)||4||12||13.2 |- | AmpR6.2Sol (10uM)||4||12||13.2 |- | 50X SYBG I||0.8||2.4||2.64 |- | H2O||31.2||93.6||102.96 |- | |} [[Image:11_7_09_QPCR.jpg]] ==11/10/09== *Run a TBE Gel for all amplified products *Gel Setup (2 10well gels) L|50J|50G|50N||30J|30G|30N|| L|10J|10G|10N||6J|6G|6N|| {| {{table}} |||Template||6X Laoding Dye||H2O |- | J||5||5||5 |- | G||5||5||5 |- | N||5||5||5 |- | |} *gel stain issue, very strong band covering everything, repeat with new setup ==11/11/09== *Repeat qPCR, stop when samples reached plateau [[Image:20091110_172133.jpg]] *Run a TBE gel with 1KB ladder [[Image:ZhangLab_2 2009-11-11 16hr 57min.jpg]] *something wrong with the 1KB ladder, huge smear indicates over amplification *Repeat qPCR stop 3 cycles before it ends ==11/12/09== {| {{table}} | ||1well (ul)||X 8 ||X1.1 |- | Template||10|||| |- | 2X Phusion||50||400||440 |- | AmpF6.2SoL (10uM)||4||32||35.2 |- | AmpR6.2Sol (10uM)||4||32||35.2 |- | 50X SYBG I||0.8||6.4||7.04 |- | H2O||31.2||249.6||274.56 |- | |} 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 20-> 72C 5 min -> 15C hold.
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