what is your reaction system of digesting DNA? - (Oct/28/2005 )

I need to digest my plasmid PK18mob with BamH I and EcoR I(from Promega),my reaction system is:

50ul PK18mob(dense);
10ul BamH I;
10ul EcoR I;
20ul Muti-Co buffer;
10ul ddH2O;

could you give any suggestions about my reaction system?and how much enzyme do you often use in your system of digesting DNA? how much buffer ?and what is your principle?Thank you in advance.

IS your buffer 10X, that is usually the case. Also, BamHI and EcoRi are compatable enzymes so it is okay to use the same buffer. How much DNA (ug) are you digesting? That will determine how many units of enzyme to use. In most commercial enzymes, 1-2 ul is more than enough to digest several ug of supercoiled DNA.

Yeah, 10uL of each enzyme is pretty excessive. Also, the way you listed the reaction has 20% of the volume consisting of your enzymes. Since most restriction enzymes come in some glycerol you end up having problems with star activity if you exceed 10% of your reaction volume w/ enzymes. Depending on how much DNA you're digesting either scale back the amount of enzymes or increase the volume of your reaction.

-Hank

thank you all the time.yes, i know there is a problem in my way,but i wanna know how to decide the proportion of components during enzyme digesting.

Check how many units there are in one µl (they usually come quite concentrated, if you have a lot of time, one or 2 µl is enzyme is enough for me). Also (as Harish said), don't go too high on glycerol, it is a cause of star activity. If you really need that much enzyme: increase your reaction volume.

Like vairus said check the conc of enzyme. Also you dont need so much enzyme if your DNA is not very concentrated, you can just leave it for longer time.. And your buffer conc is less than 10 X is it? From your reaction mix it looks like you're adding quite a lot for a 100 microlitre reaction, if i read that right.

I'd strongly recommend that you read the section "setting up restriction digests" at the NEB site, here:
http://www.neb.com/nebecomm/tech_reference...up_reaction.asp

And consult the double digest calculator here:
http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

As others have said, you have too much enzyme for that volume; also, I don't know what the buffer is, but it probably is 10x not 5x; also, you have given us no idea on the DNA concentration. Think twice about reacting more than about 2 ug in a 100 ul volume. Also, depending on the buffer the DNA is in, the large volume of DNA added to the reaction could impact the buffer behavior. I would be trying to keep the DNA volume added to less than 25% of the reaction volume. If you need to concentrate the DNA, do an ethanol precipitation before hand.

thank for your help!now i catch some things about enzyme digesting,but what is "star activity"?

Star activity is basically when a restriction enzyme cuts at sites other than it's usual recognition site.

-Hank