restriction problem with pMON999

Bryan Maloney bjm10 at cornell.edu
Thu Sep 6 23:30:32 EST 2001


Ivo Rieu <irieu at sci.kun.nl> wrote in news:3B938D75.BA5C3EA4 at sci.kun.nl:

> Hello,
> For a long time, I have been working to clone plant receptor-kinase
> cDNAs fused to GFP cDNA in pMON999. Many, many times my minipreps are
> not susceptable to restriction enzymes, in other words, they cannot be
> cut. But sometimes they can be cut! Does this sound familiar. In my
> other cloning work, with different cDNAs in different vectors
> (pBlueScript, pGEM etc.) I never encounter this problem. Please, share
> any thoughts with me...
> I tried adding glucose to silence the LacZ promoter. If it is leakage
> from my 35S-CMV promoter, how can it be so variable and what can I do to
> overcome the problem?

We used to have that problem, especially with Agrobacterium preps, until we 
switched miniprep methods.  Our method:

pellet 1.5-3 ml culture (3ml for agrobacterium).
Resuspend in 50 ul TE
Add 300 ul of "TENS" (0.2M NaOH, 0.1% SDS, in TE)
Incubate 5minutes RT
Add 200 ul of 7.5M ammonium acetate
Incubate 15 minutes on ice.
Spin 10 minutes at four degrees in microcentrifuge.
Add 330 ul isopropanol
Incubate 10 minutes RT
Spin 10 minutes RT
Wash pellet with 70% ethanol 
Resuspend in TE.

Be sure that TE is the basis of the "TENS", not water.  Having accidentally 
diluted the NaOH and SDS stocks in water, it just doesn't work as well.

There will be a good deal of RNA in the preps.  If this is a problem, add 
RNAse to the supernatant and incubate 10-15 minutes at RT before adding 
isopropanol.

Waving a rattle and doing a little dance doesn't hurt, either.


-- 
"Cape Cod Salsa--somehow that's just not right."




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