Revision as of 21:12, 30 December 2009 by >Alan6017518
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
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