Recommended Protocol for Methylation of Genomic DNA

1. Dilute supplied SAM (32 mM stock solution) to 1600 µM 
   
   SAM (32 mM stock solution) 1 µl
   Nuclease-free Water 19 µl
   
   
2. Reaction set-up (add in the following order):
   
   

   Nuclease-free Water	up to 50 µl
   Supplied Methyltransferase Reaction Buffer (10X)	5 µl
   Diluted SAM (1600 uM)	5 µl
   DNA	1 µg
   Methyltransferase	4 – 25 units (1 µl)

3. Mix, Pipette up and down at least six times
   
   
4. Incubate at 37°C for 1 hour
   
   
5. Stop the reaction by heating at 65°C for 20 minutes

Notes:

1. 4 - 25 units (1 µl) methyltransferase/µg of DNA is recommended.
   
   
2. The volume of DNA should be 25% or less of the total rxn volume. Adjust the reaction total volume accordingly. Having too much volume of substrate DNA in the reaction can cause inhibition by changing the pH or salt concentration of the reaction.
   
   
3. Up to 4 µg of purified, clean DNA can be methylated in a 20 µl reaction. The SAM concentration should be adjusted to 640 µM.
   
   
4. The incubation time can be increased to 4 hours. Overnight incubations do not give significant increases in methylation.
   
   
5. The above protocol can also be used for other types of DNA including plasmids and purified PCR products.