Methyltransferase Assay .
Set up the assays , to contain 50.0 µL total per assay with 3 µg of DNA and 2 µCi of Adenosyl - L - Methionine , S - Methyl ( 3H ) , and the enzyme to be tested .
Incubate the assays at 37°C for 2 hours .
Add 30 µL ( 60 µg ) of proteinase K to each assay .
Incubate at 65°C for 20 min .
Add 4 µL ( 20 µg ) of ss salmon sperm DNA as a carrier .
Add an equal volume ( 84 µL ) of 20 % TCA and incubate at 4°C for 10 min to precipitate the samples .
Centrifuge the assays in the microfuge for 5 min at 4°C .
Discard the supernatants .
Wash the pellets 2X with 200 µL of 5 % TCA in the microfuge for 5 min at 4°C .
Resuspend the pellets with 500 µL each of 500 mM NaOH .
Incubate the assays at 65°C for 20 min .
Precipitate the samples with 500 µL of 20 % TCA for 10 min at 4°C .
Centrifuge the samples in the microfuge for 5 min at 4°C .
Wash the pellets 2X with 200 µL of 5 % TCA in the microfuge for 5 min at 4°C .
Resuspend the pellet with 500 µL of 5 % TCA .
Incubate in boiling water for 30 min ( loosen the caps or puncture the caps of the tubes ) .
Centrifuge the samples in the microfuge for 5 min at 4°C to remove the insoluble material .
Count the supernatants in 10 mL of triton - toluene counting solution .
