New England Biolabs, B9003S, S-adenosylmethionine (SAM)

Related Categories Restriction Endonuclease Buffers & Diluents,, Methyltransferases for Epigenetics,, Buffers Applications Restriction Enzyme Digestion FAQ Q: What is the buffer composition of your S-adenosyl-methionine (SAM)? A: The SAM is prepared in a solution of 0.005M H2SO4 (sulfuric acid) and 10% ETOH. The pH is 4.0 at 25 °C. Q: Will the buffer from the SAM inhibit my reaction? A: The concentration of SAM is 32 mM. Most often it is used at 80 μM which is a 1:400 dilution. At this level the SAM buffer has little effect on subsequent reactions. SssI Methylase shows full activity at SAM concentrations of 320 μM, a 1:100 dilution. Q: What quality controls are performed on your SAM? A: Each lot of SAM is tested in an SssI (CG) Methylase (NEB# M0226) reaction with Lambda DNA (NEB# N3011). The DNA is then challenged with BstUI (NEB# R0518) to confirm methylation. Q: Is SAM supplied with enzymes that require it in the reaction buffer? A: SAM is provided with all enzymes that require it. It can also be ordered separately as product NEB# B9003S (5 vials of 100 μl each of a 32 mM solution). Q: How stable is SAM in a reaction? A: SAM is unstable at pH 7.5, 37 °C and should be mixed fresh prior to starting the reaction. Q: Should I replenish the SAM in my reaction? A: SAM is unstable at pH 7.5, 37 °C. For extended reactions adding more SAM after 4 hours can improve results. Methylation reactions, however, are also greatly affected by S-adenosylhomocysteine. S-adenosylhomocysteine, the byproduct of the methylation reaction binds more tightly to methylases than does SAM. Inhibition by S-adenosylhomocysteine greatly reduces the reaction rate as time passes. Using more enzyme in shorter reactions may improve methylation.