IGEM:IMPERIAL/2009/M3/Assays/Meth
Dam Methylation Assay
Assay
This assay is to indirectly measure the activity by Dam methylases. We use DpnI as it only cuts DNA at a methylated site.
The same principles are used for restriction enzyme digest assay.
Protocol
Materials:
- Oligos A, B and C for Dam methylase tagged with TAMRA and Dabcyl
- Buffer:
100 μl 20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 100 μg ml−1 bovine serum albumin (BSA)
- Purified DpnI
- S-(5′-Adenosyl)-L-methionine p-toluenesulfonate salt (SAM)
Procedure
1) 0.125, 0.25, 0.5, 1, 1.5 and 2 μM of substrate were diluted in buffer.
2) Dam methylase purified from cell extract was added at a concentration which was at least 10-fold lower than the substrate concentration.
There was a control setup where no Dam methylase was added.
3) The buffer, probe substrate, 160μM SAM and 8 units of Dpn I were added into spectrophotometric cuvettes in order.
4) Samples were equilibrated to the incubation temperature, which was confirmed by a steady background fluorescence emission over 5 minutes.
5) Dam methylase purified from cell extract was added to initiate methylation.
6) At defined time intervals, the average fluorescence at the wavelengths indicated above were determined. (The excitation wavelength was 560 nm for TAMRA, while the emission was measured between 570 and 590 nm for TAMRA in 0.2 nm steps).
7) To complete substrate cleavage a 10-fold excess of enzyme was added at the end of the experiment.
8) Initial velocities (v0) were calculated from the linear part of individual reaction progress curves.
