Question about DM1 bacteria strain.

jh7 at acsu.buffalo.edu jh7 at acsu.buffalo.edu
Mon Mar 10 09:38:00 EST 1997


Hi there:

I have problems with extracting plasmid from DM1(dam/dcm negative strain).
If you know the answer, please help me out. Thanks!

I am trying to cut my plasmid with Bcl I witch requires the DNA to be
isolated from dam negative bacteria strain. So I transformed the DM1
bacteria which was provided from Gibco. It should be both dam and dcm
negative. 

My plasmid contains the pCAT Baisc and a promoter region in front of it.
So I spread the transformed cell on Amp plates. It seems the
transformation was good, there were normal colonies on the Amp plate after
transformation. So I chose several single colonies and cultured and did
the mini-prep. 

Then came the problem. There was no DNA at all. I did three times with
protocal used routinly in our lab(alkaline lysis followed by phenol/chloroform
extraction). I did see pellet but it turned out to be RNA( low M.W.
smear band on agrose gel). 

I did control for the DM1 competent cell with water transformation, also
checked the Amp plate and liquid medium. They are all good.

Can anyone give me a clue?

Thanks!

yuan
  




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