I would like to understand what volume of SAM is ideal for RFLP using the recommendation for use of the methylation reaction where the SAM should be diluted 1:400 to a final concentration of 80μM.
As far as I understand your question, the answer is you do not need to add SAM to RFLP mix.
Thank you Agata, the issue is that the HeaIII restriction enzyme shipped to our lab was supplied with buffer and S-adenosyl-methionine (SAM). The protocol for the use of SAM was not specifically stated as it was rather vocal about the concentration of the SAM and silent on the volume i.e. HaeIII methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X HaeIII methyltransferase reaction buffer, supplemented with 80 µM SAM
What is the concentration of SAM? You can for sure find such information in the datasheet.
S-adenosyl-methionine (SAM) is included with all of the DNA methylases available from company. As well as certain restriction enzymes for which it is required. The SAM is prepared in a solution of 0.005 M H2SO4 and 10% ETOH and filtered. 1.For use of methylation reaction the SAM should be diluted 1:400 to a final concentration of 80μM
One ot two microliters of S-adenosyl-methionine (SAM) is usually a convenient pippeting volume into a 20 ul enzyme reaction. If the final SAM concentration is to be 80 uM it follows that its concentration in a stock solution should be 400-800 uM. Please, bear in mind that SAM is inherently iunstable due to hydrolysis. It should be stored at high concentration (>10 mM) at -20C.
Are you sure you've bought the endonuclease and not the methylase? HaeIII endonuclease does not require SAM for activity.
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