Method I
A. Methylation
Combine in a total volume of 50 µl
- Restriction endonuclease reaction
buffer (used for methylation and subsequent cleavage)
- 1-2 µg DNA
- 80 µM SAM* (supplied as 32 mM SAM, 5 mM H2SO4, 10% EtOH)
3-10 units Methylase
Incubate 1 hour at 37°C
Incubate 15 minutes at 65°C (to inactivate methylase)
B. Cleavage
Add 2 units restriction endonuclease
- Incubate 1 hour 37°C
- Analyze by agarose gel electrophoresis
Method II
If a protocol which recommends EDTA is used in the methylation reaction, special
care must be taken to avoid the chelation of Mg++ by the EDTA in the cleavage
reaction. The following protocol addresses the problems associated with an
EDTA containing methylase buffer.
A. Methylation
Combine in a total volume of 10 µl:
- EDTA-containing methylase buffer
- 1-2 µg DNA
- 80 µM SAM* (supplied as 32 mM SAM, 5 mM H2SO4, 10% EtOH)
- 1-5 units Methylase
Incubate 1 hour at 37°C
B. Terminate Methylation
- Heat to 65°C for 10 minutes
- Add 40 µl restriction endonuclease assay buffer containing 20 mM Mg++
- Proceed to Cleavage, Step C
or
- Add 50 µl 10 mM Tris-HCI (pH 7.5), 0.2 M NaCI
- Add 50 µl phenol
- Phenol extract and collect aqueous (top) layer
- Precipitate with 100 µl isopropanol at -20°C (minimum 20 minutes)
- Pellet DNA and wash gently with 100% isopropanol
- Discard isopropanol and air dry pellet
- Resuspend pellet in 50 µl restriction enzyme buffer
C. Cleavage
- Add 5-10 units restriction endonuclease
- Incubate 1 hour 37°C
- Analyze by agarose gel electrophoresis
* S-adenosylmethionine (SAM) is
supplied with each vial of methylase
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