Is there a simple test for RNase and DNase free

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Mon Jan 29 12:28:24 EST 2001


In article <6lhd6.14135$KP3.4431209 at news3.rdc1.on.home.com>, the eminent
EO at Excite at Home - The Leader in Broadband http://home.com/faster wrote
>Can you be more specific as to how much buffer and suspect to use?  How
>about 50 mL buffer with 0.1 gram reagent.?

That is a little OTT! 

1ug of a Hae III digest of pBR322 (or similar) should be trashed with
less than a fraction of a unit of DNAse. As pure DNase is probably
3000u/mg then I would hazard a guess that ng quantities of DNase would
be picked up, maybe even pg. Easy to test. 

Prepare a reaction mix with 1ug of digested plasmid/50ul and dispense
45ul into 10 microtubes. 

Prepare a 2u/ul stock of DNase in same buffer. 

Add 5ul (10u) DNase to tube 1. Mix. Remove 5ul with a fresh tip to tube
2. Mix and repeat down the line remembering not to add DNAse to last
tube 

1st tube has 0.2u DNase/ul
2nd tube 0.02
3rd 0.002 etc etc

Incubate 37C for one hour.

Run on acrylamide gel, stain with EtBr and see what happens. For
increased sensitivity 37C incubation can be O/N or weekend etc. Further
increase using 1/10 amount of DNA and use Sybr Gold for staining etc.
etc.

If you want to test larger plastics then use say a 12ml volume, shake it
all around the plastic, spin it down and transfer to known clean sterile
plastic tube for the incubation.

Basically you have to be a little inventive.

Duncan  
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






More information about the Methods mailing list