3 replies to this topic

[squallweathered]

Hi all,

 

Apologies, I should know this but I am rusty on enzymology.

 

Having an issue where we have an enzymatic reaction (type of restriction digest of DNA in this case) that performs fine at full volume. When we keep the enzyme, substrate, buffer concentrations the same but reduce the overall reaction volume, the activity of the enzyme (measured in this case in the emergence of smaller DNA fragments) appears to increase.

 

Is there a reason for this - volume based change in the reaction kinetics?

 

Many thanks

[mdfenko]

is it concentration of enzyme and substrate or amount added that are kept constant?

 

if amount then you have a higher concentration, resulting in greater activity.

 

if concentration then the greater activity could be due to greater uniformity and more rapid equilibration of assay conditions with lower volume.

[phage434]

Small volumes can sequester DNA or protein or both on the side walls of tubes. Perhaps this is the issue here.

[squallweathered]

Many thanks mdfenko and phage434.

 

 

is it concentration of enzyme and substrate or amount added that are kept constant?

 

We scale down the added volumes by 20x of all reagents, so the amount added is reduced, but the concentration of each relative to each other is maintained.

 

Thanks for all advise.