AlanFung:LabNotes/Capturing/CPG97K Low-input/2009-10-24: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 53: | Line 53: | ||
==Capturing System Setup== | ==Capturing System Setup== | ||
*Prepare total volume up to 15ul | *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 | |||
|- | |||
| | |||
|} | |||
CT Converted Ms-gDNA | CT Converted Ms-gDNA |
Revision as of 17:14, 3 November 2009
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 Nanodrop:
RNA
Capturing System Setup
- Prepare total volume up to 15ul
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 | |
CT Converted Ms-gDNA 10X AmpLigase buffer Ms_Meth Probe (242.4nM) H2O Total 95c 10min -> 60C 24h ->add 2ul SLN mix-> 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