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