4 replies to this topic

[proteinz]

This is a really stupid question but when you are adding a restriction enzyme to digest DNA do you add enzyme to give a final concentration of x units in your final volume or simply add the required units into the reaction?
I am confused!

[Ameya P]

proteinz, on 14 January 2011 - 04:22 AM, said:

This is a really stupid question but when you are adding a restriction enzyme to digest DNA do you add enzyme to give a final concentration of x units in your final volume or simply add the required units into the reaction?
I am confused!

Its not a stupid question...I think it is added to give a final concentration of x units in the final volume.... but usually it is 1ul/ 2ul of RE was used to digest the sample volume of x ul

[lchihu]

It´s not a stupid question. By definition 1 Unit of enzyme is used to digest 1ug of DNA (Lambda) in a 50ul reaction in 1 hour.
Best regarda

[leelee]

Add the required units to the reaction.
It is independent of volume- provided you use a realistic volume of course (for example, 1000ul would be silly!) and you must also make sure that the volume of RE you add doesn't exceed 10% of the reaction volume.

I typically use 1ul of enzyme in anything from 15ul up to 50ul depending on my application.

*edited to add this link to NEB for a good source of info for RE
http://www.neb.com/n...tegory1.asp#661

Edited by leelee, 23 January 2011 - 10:44 PM.

[proteinz]

thanks all for your help! i will just add the required units then!
have a nice day