AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 2: Difference between revisions
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==Bisulfite Conversion of Sample NA12878,NA18507, NA20431, CV-Fibr, CV-iPS-B, CV-iPS-F w/+ve and -ve Control== | |||
==Overview== | ==Overview== | ||
Line 7: | Line 5: | ||
*Sample Digestion with Proteinase K | *Sample Digestion with Proteinase K | ||
*Bisulfite Conversion of DNA | *Bisulfite Conversion of DNA | ||
==Sample== | |||
NA12878 (160ng/ul) | |||
NA18507 (257ng/ul) | |||
NA20431 (314ng/ul) | |||
CV-Fibr (712ng/ul) | |||
CV-iPS-B (358ng/ul) | |||
CV-iPS-F (309ng/ul) | |||
==Sample Preparation== | |||
{| {{table}} | |||
| Sample Prep||(ng/ul)||To Get 1500 ng (uL)||Make it up to 60ul | |||
|- | |||
| NA12878||160.0||9.4||50.6 | |||
|- | |||
| NA18507||257.0||5.8||54.2 | |||
|- | |||
| NA20431||314.0||4.8||55.2 | |||
|- | |||
| CV-Fibr||712.0||2.1||57.9 | |||
|- | |||
| CV-iPS-B||358.0||4.2||55.8 | |||
|- | |||
| CV-iPS-F||309.0||4.9||55.1 | |||
|- | |||
| | |||
|} | |||
==Reagent Preparation== | ==Reagent Preparation== | ||
===CT Conversion Reagent (1250ul Total good for 9rxns)=== | |||
===CT Conversion Reagent=== | |||
*CT Conversion Reagent is light sensitive, so minize its exposure to light | *CT Conversion Reagent is light sensitive, so minize its exposure to light | ||
*Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent | *Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent | ||
*Mix by frequent vortexing at RT for 10m | *Mix by frequent vortexing at RT for 10m | ||
*Mix 160ul of M-Reaction Buffer and mix an additional 1m | *Mix 160ul of M-Reaction Buffer and mix an additional 1m | ||
===M-Wash Buffer=== | ===M-Wash Buffer=== | ||
*Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use | *Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use | ||
Line 21: | Line 46: | ||
==Protocol== | ==Protocol== | ||
==For optimal results use 500ng of DNA per treatment== | |||
==For optimal results use 500ng of DNA per treatment (3 treatments for each sample)== | |||
===Section II Bisulfite conversion of DNA=== | |||
{| {{table}} | {| {{table}} | ||
| | | Sample||Volume||CT Conversion Reagent||Treatment | ||
| | |- | ||
| | | NA12878||20.0||130.0||x3 | ||
|- | |||
| NA18507||20.0||130.0||x3 | |||
|- | |||
| NA20431||20.0||130.0||x3 | |||
|- | |- | ||
| | | CV-Fibr||20.0||130.0||x3 | ||
|- | |- | ||
| | | CV-iPS-B||20.0||130.0||x3 | ||
|- | |- | ||
| | | CV-iPS-F||20.0||130.0||x3 | ||
|- | |- | ||
| | | Jurkat||20.0||130.0||x1 | ||
|- | |- | ||
| | | Blank (ddH2O)||20.0||130.0||x1 | ||
|- | |- | ||
| | | | ||
|} | |} | ||
* | *Add 20ul of sample to 130ul of CT conversion reagent solution in a pcr tube | ||
*Vortex the sample to mix | *Vortex the sample to mix | ||
*Pulse centrifuge | *Pulse centrifuge |
Latest revision as of 21:12, 30 December 2009
Bisulfite Conversion of Sample NA12878,NA18507, NA20431, CV-Fibr, CV-iPS-B, CV-iPS-F w/+ve and -ve Control[edit]
Overview[edit]
- Sample preparation
- Sample Digestion with Proteinase K
- Bisulfite Conversion of DNA
Sample[edit]
NA12878 (160ng/ul) NA18507 (257ng/ul) NA20431 (314ng/ul) CV-Fibr (712ng/ul) CV-iPS-B (358ng/ul) CV-iPS-F (309ng/ul)
Sample Preparation[edit]
Sample Prep | (ng/ul) | To Get 1500 ng (uL) | Make it up to 60ul |
NA12878 | 160.0 | 9.4 | 50.6 |
NA18507 | 257.0 | 5.8 | 54.2 |
NA20431 | 314.0 | 4.8 | 55.2 |
CV-Fibr | 712.0 | 2.1 | 57.9 |
CV-iPS-B | 358.0 | 4.2 | 55.8 |
CV-iPS-F | 309.0 | 4.9 | 55.1 |
Reagent Preparation[edit]
CT Conversion Reagent (1250ul Total good for 9rxns)[edit]
- CT Conversion Reagent is light sensitive, so minize its exposure to light
- Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent
- Mix by frequent vortexing at RT for 10m
- Mix 160ul of M-Reaction Buffer and mix an additional 1m
M-Wash Buffer[edit]
- Add 24ml of 100% ethanol to the 6ml M-Wash Buffer concentrate (D5020)before use
- Add 96ml 100% ethanol to the 24ml M-Wash buffer concentrate (D5021) before use
Protocol[edit]
For optimal results use 500ng of DNA per treatment (3 treatments for each sample)[edit]
Section II Bisulfite conversion of DNA[edit]
Sample | Volume | CT Conversion Reagent | Treatment |
NA12878 | 20.0 | 130.0 | x3 |
NA18507 | 20.0 | 130.0 | x3 |
NA20431 | 20.0 | 130.0 | x3 |
CV-Fibr | 20.0 | 130.0 | x3 |
CV-iPS-B | 20.0 | 130.0 | x3 |
CV-iPS-F | 20.0 | 130.0 | x3 |
Jurkat | 20.0 | 130.0 | x1 |
Blank (ddH2O) | 20.0 | 130.0 | x1 |
- Add 20ul of sample to 130ul of CT conversion reagent solution in a pcr tube
- Vortex the sample to mix
- Pulse centrifuge
- Perform
*98C for 8m *64C for 3.5hr *4C hold
- Add 600ul of M binding buffer into a column assembly
- Load sample(s) to the column
- Close the cap and mix by inverting the column several times
- Centrifuge at >10,000g for 30sec
- Discard the flow through
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
- Centrifuge for 30sec
- Place the column into a 1.5ml tube
- Add 10ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements)
- Centrifuge for 30sec to elute the DNA