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V17

22.1.17

Bisulfite conversion and MSP Protocol
Bisulfite conversion kit - EZ DNA methylation-Gold kit (Zymo)
- If using EZ methylation lightning kit (Preferred) -Use the kit's manual as it is (page 5).
A) Bisulfite conversion reagent Preparation
(use provided Bisulfite conversion reagent Tube) PROTECT FROM LIGHT
1. Spin down briefly CT-conversion Reagent Tube.
2. Add to CT-conversion Reagent Tube:
a. 900µl H2O
b. 300µl of M-Dilution Buffer
c. 50 µl M-Dissolving Buffer
3. Vortexing for 10min at RT. Or vortex every minute to a few seconds.
CT conversion reagent – use immediately or store 1 week at 4º, one month at -20º.
If stored: heat to 37º and vortex before use.
B) 1 µg of DNA in 45 µl H20 + 5 µl M-dilution buffer.
37º for 15min
C) Add to the 50 µl DNA sample tube 100 µl of Bisulfite conversion reagent.
* The Bisulfite conversion reagent can be good for about 12-13 sample, not just 10 as
specified in the manual.
D) In Thermal cycler perform (original kit version):
98ºC for 10 min
64ºC for 2.5h
4º (up to 20 h)
Or use instead:
Alternative method: (Use this one).
95ºC for 30 sec, 50ºC for 15min
->
20 cycles
4º (up to 20 h)
Total time – 5 Hours
E) Purification (Modified methylation protocol):
1. Put samples on Ice – 15Minutes.
2. Load 400 µl M-binding buffer and after that the sample (all the sample) into
Zymo-spin I column.
3. Mix by inverting the column+tube several times.
4. Centrifugate >10,000 g for 30sec. Discard flow.
5. Add 200 µl M-wash buffer and centrifugate (30sec)
6. Add 200 µl M-desulfonation Buffer
7. Wait 15min
8. Centrifugate for 30sec, discard flow
9. Add 200 µl M-wash buffer and centrifugate (30sec)
10. Add 200 µl M-wash buffer and centrifugate (1min)
11. Place the column in a new 1.5ml tube. And write the sample name on the tube.

12. Elute material with 50µl H2O.
( not as specified in the manual with 10µl M-elution, this way you will gain more
material volume).
Centrifugate for 30sec.
13. Store converted DNA at -80oC.
F) Take 2.5 µl of converted DNA for PCR
- Perform a PCR step for each sample with a methylated and un-methylated primers in
different tubes separately, and NTC.
* PCR conditions are dependent on your enzyme, primers and samples.
PCR conditions (Gotaq hot start PCR kit):
95˚C for 10 min – 1 Cycle
94˚C for 20 sec, 66˚C for 30 sec, and 72˚C for 1min – 35 Cycles
72° 10 min – 1 cycle
4˚C - ∞
Total 77 Minutes
-----------------------------------------------------------------------------------------------------------MSP (Methylation Specific PCR)
:Background
If there is a methyl group (H3C) on a C base in the DNA in CpG (Cytosine-Phosphate- Guanine) areas, it can effect gene expression, mostly the gene will be less accessible to
.transcription factors, and will be silenced
Bisulfite conversion (CT conversion):
The system is based upon converting all unmethylated cytosine residues to uracil using chemical treatment. The technique consists of treating DNA with bisulfate, which
converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged
Than you can design and use specific primers to methylated an non methylated areas on .the NDA
.Run the product in agarose gel, Or use 3500XL genetic analyzer -

Written by: Itai, Elad