AlanFung:LabNotes/Capturing/Ms Meth/NIH 3T3 Ms gDNA/2009-6-5
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Objective[edit]
- Prepare two tubes of bisulfite converted Ms and Jurkat gDNA enough for plenty of experiments
- Perform capturing with MS_Meth probes and Alice's Probes for positive control
- Follow closely to Ida's protocol
Overview[edit]
- Bisulfite Conversion of DNA
- Capturing Reaction
Reagent Preparation[edit]
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
Procedures[edit]
Bilsulfite Conversion with EZ DNA Methylation-Gold Kit[edit]
- 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:
*Ms_gDNA (CT Converted) 384.7ng/uL * 10uL *Jurkat_gDNA (CT Converted) 368.7ng/uL * 10uL
*Dilute Ms_gDNA (CT Converted) with 7.85uL ddh2o to 200ng/uL *Dilute Jurkat_gDNA (CT Converted) with 7.17uL ddh2o to 200ng/uL
Calculation for Ms_Meth Probe Set[edit]
Probe:target ratio 200:1 NIH_3T3_gDNA CT Converted Template(200ng/uL) Use 200ng total, so = 1uL
- 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
Calculation for CPG30K probe Set[edit]
Probe:target ratio 200:1
Jurkat_gDNA CT Converted Template(200ng/uL)
Use 200ng total, so = 1uL
- 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*30,000
CPG_30K 10ng/uL 30,000Probes 100nt Since 1 base = 330Da (for single stranded DNA, 660Da for ds), the concentration of probe is 10 ng/ul /(108x330) = 0.000280584 = 280.58nM # 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 30,000 / [(6x10^23)x (280.58*10^-9M)]*10^6 =2.37uL
Capturing System Setup[edit]
CT Converted Ms-gDNA (200ng/uL) 1uL 10X AmpLigase buffer 1uL Ms_Meth Probe (242.4nM) 1.83uL H2O 6.17uL Total 10uL 95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h ->
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
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
*Noy enough SLN mix, added less than 2uL for the Jurkat DNA capturing reaction
PCR with AmpF/R6.2Sol and iProof[edit]
TubeA TubeB Jurkat DNA Template 0uL 5uL Ms DNA Template 5ul 0uL 2X iProof Mastermix 50ul 50uL AmpF6.3SoL (10uM) 4ul 4ul AmpR6.3SoL (10uM) 4ul 4ul 50X SYBG I 0.4ul 0.4ul H2O 36.6ul 36.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
- Ms DNA taken out one cycle earlier
TBE Gel Run[edit]
- 0.5uL low mass ladder + 3uL 6X gel loading dye
- 3uL amplified Jurkat DNA + 3uL 6X gel loading dye
- 3uL amplified Ms DNA + 3uL 6X gel loading dye
File:ZhangLab 2 2009-06-09 14hr 58min crop.jpg
- Ms DNA seems to be over amplified
- Rerun another PCR amplification for Ms DNA template only (-1 cycle)
TubeA Ms DNA Template 5ul 2X iProof Mastermix 50ul AmpF6.3SoL (10uM) 4ul AmpR6.3SoL (10uM) 4ul 50X SYBG I 0.4ul H2O 36.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 8-> 72C 5 min -> 15C hold.
File:ZhangLab 2 2009-06-09 17hr 28min-2 crop.jpg
- Gel band is more concentrated around 400bp, however there is still a smear, may be due to capturing of varying length of targets.