restriction problem with pMON999

Ivo Rieu irieu at sci.kun.nl
Mon Sep 3 09:02:29 EST 2001


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?

My prep method:
pellet 2 ml culture (DH5a in LB-amp)
100 ul TEG, vortex to mix well
200 ul NaOH (0.2M) SDS (1%), invert, 2 minutes room-temp
150 ul KAc (5M, pH4.8), invert, 2 minutes room-temp
centrifuge 5' room-temp
precipitate with 0.8 ml EtOH, 2 minutes room-temp
centrifuge
wash with 70% EtOH, 2 minutes room-temp
centrifuge
in 50 ul TE with 20 ug/ml RNase

Digestion:
2 ul DNA
1.5 ul buffer
0.5 ul enzyme
11 ul water
2h 37 degr. Celcius

Seperate on 1% agarose gel







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