Recommended Protocol for Methylation of Genomic DNA
- Dilute SAM to 1600 μM using the supplied 32 mM stock.
a. SAM 32 mM stock 1 μl
b. Nuclease free water 19 μl
- Add in order:
a. Nuclease free water 14 μl
b. 10X NEBuffer 2 2 μl
c. Diluted SAM from step 1 2 μl
d. Genomic DNA (1 μg) 1 μl
e. SssI methylase (4 U/μl) 1 μl
- Mix, Pipette up and down at least six times.
- Incubate one hour at 37°C.
- Stop the reaction by heating at 65°C for 20 minutes.
1. The volume of DNA can be increased to 5 μl. When using more dilute DNA increase the reaction volume to 50 μl. Using too much DNA volume in the reaction can cause inhibition by changing the pH or salt concentration of the reaction.
2. Up to 4 μg of DNA can be methylated in a 20 μl reaction. The SAM concentration should be adjusted to 640 μM. Concentrated SssI M0226M (1 μl of 20,000 U/ml) should be used.
3. The incubation time can be increased to 4 hours. Overnight incubations do not give significant increases in methylation.
4. The above protocol can also be used for other types of DNA including plasmids and purified PCR products.
Example of a large-scale methylation:
1. Add in order:
a. Nuclease free water 220 μl
b. 10X NEBuffer 2 50 μl
c. SAM 32 mM 10 μl
d. Lambda DNA (500 μg/ml) 200 μl
e. SssI methylase (20 U/μl) 20 μl
2. Mix, Pipette up and down at least six times.
3. Incubate 2 hours at 37°C.
4. Stop the reaction by heating at 65°C for 20 minutes. The DNA can be purified by phenol extraction followed by ethanol precipitation or by using a commercial DNA purification kit. For long-term storage at -20°C, suspend in TE.