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Buffer Substitution: Calcium for Magnesium

buffer magnesium calcium

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4 replies to this topic

#1 hotstuffdb22

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Posted 04 January 2014 - 03:15 PM

Hello!

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

100mM NaCl

30mM MgCl2

 

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?

 

Thanks!



#2 hobglobin

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Posted 05 January 2014 - 04:12 AM

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.



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#3 hotstuffdb22

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Posted 05 January 2014 - 04:18 AM

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?



#4 hobglobin

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Posted 05 January 2014 - 08:39 AM

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.



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#5 hotstuffdb22

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Posted 18 January 2014 - 04:57 AM

Thank you very much!  The simple substitution worked to perfection!







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