Buffer Substitution: Calcium for Magnesium - (Jan/04/2014 )
Any help with the question below will be much appreciated! Thanks in advance!
I have a buffer I use for digesting DNA. It is composed of the following:
100mM Tris-HCl pH 7.4
I want to remove magnesium in this assay and substitute for calcium. Thus, can I simply use 30mM CaCl2 instead of 30mM MgCl2? Or do I need to consider the difference in molecular weight between calcium and magnesium and adjust the final concentration?
As far as I remember more or less all a restriction enzymes, nucleases/DNAses need Mg2+ as cofactor for enzyme activity. Therefore if you replace it with Ca2+ an enzymatic digest won't work.
Thanks a lot for the message.
I'm actually trying to inhibit the activity of the enzyme, so I want to substitute with calcium. Can I simply substitute 30mM CaCl2 for the MgCl2, or do I need to adjust the NaCl and Tris at the same time?
The molarity and ionic strength would be the same if you replace them, also pH would not change as you have a Tris buffer (if you leave the concentration as it is). But I've no idea what important is for your experiment.
Thank you very much! The simple substitution worked to perfection!