Biochemical S.O.S - (Jul/03/2009 )

Well I have a recombinant enzyme, it is an endonuclease and when you try it out with plasmid DNA and run it on a gel it degrades the DNA. I need to run some biochemical tests and find son biochemicals values like km and stuff, but I'm totally lost I dont know how to begin and what to do, this is the real biochemical hard core stuff, the cloning, expression and purification was a piece of cake...I don't even know how to determinate an unit (U) of a recombinant enzime.

Heeeeeeelp!

And yes, english is not my native languaje

-hineiko-

Hey,

Do you run a control with no endonuclease and see that he DNA is actually there. The degradation could be because of some other factor. Also, if you could paste the gel pic, it would be helpful. Hope you are using the right proportions.

Best,
TC

hineiko on Jul 3 2009, 09:59 PM said:

-T C-

T C on Jul 4 2009, 04:10 PM said:

hineiko on Jul 3 2009, 09:59 PM said:

Have a look here
Chapter 19 (page 287).
The definition of an u is on the instruction leaflet together with some technical reference how to do it. On the companies website is also a lot of information about this topic.

-hobglobin-

@ T C

Sure, of course I ran a control and I'm sure, as a matter of fact I ran 2 controls one without the nuclease but everything else (buffer, water) and one thats just the DNA.
1. Ladder, 2. DNA, 3. without nuclease RT, 4. with nulease RT, 5.without nuclease 37ºC, 6. with nulease 37ºC.

@hobglobin

Wow, I'm into that book like right now, thanks! great reference.

Thanks a lot, for the answers!!!

I'm pretty sure that is my enzyme the one that is degrading the DNA but since the purification was a little shitty I have to demonstrate that, running an SDS-DNA-PAGE, this forum it's pretty helpful

-hineiko-

How frequently does the enzyme cut the DNA? Did you try using some other plasmid DNA?

hineiko on Jul 6 2009, 07:31 AM said:

-T C-