problem with transformed plasmid's size - (Jun/14/2007 )

Hi,
I have a problem with my cloned plasmid. I have cloned a 4.8 kb long cDNA into a the pTARGET vector and confirmed the construct by PCR and restriction digestion. then I transformed the correct plasmid into DH5-ALPHA E. coli and growth them in LB medium and made glycerol stockes.after 1 day I used the glycerol stock and isolated plasmids show different sizes when confirming by PCR and digestion. this is repeaded for n times and the wrong size was allways the same..... now I am thinking about recombination or something like that inside the bacteri... is it possible that there is a recombination between the plasmid and cloned CDNA OR bacterial genomic DNA, OR ALSO is it possible that the EXPRESSED protein which I cloned its cDNA be responsible for such a change? I clonbed a ubiquitin specific protease into the plasmid

my money is on the nasty protease that you you have cloned into your plasmid.

Can you turn off expression?

Growing your cells at a lower temperature ~30 Celsius, and growing large volumes to extract your DNA from, could help a little.

Have you kept and are you keeping antibiotic pressure on the E. coli and how likely is a contamination with an other plasmid during your transfection?

Ah, my mistake. I thus assume your colonies were truely white when you picked them (no hint of blue). there is the lacZ gene in the pTARGET.

Anyhow, you mentioned that the restriction digest was correct. I thus gather that a miniprep was conducted to obtain that DNA. Am I correct to then assume that when a midiprep was conducted, you could not obtain your plasmid anymore.

If that is the case, I would still suggest that growing the culture at a reduced temperature would helpful. Other possible things you could do would be to use a different e coli strain.

I would agree with perneseblue's suggestion and try a different strain of bacteria or grow them for a short period of time or at reduced temp.

check your sequence ... do you have any Palindromic sequence.. ??

if you have then that part will definately be be repared by bacteria..

I used also 2 different Ecoli Strains. in the case of top-10 strain, all colonies were wrong in size but when using DH5-alpha strain, I get some correct and some wrong sizes. But in the wrong size also I dont lose the restriction sites and I confirmed a 1000 bp long fragment which is inserted into my insert (in wrong cases) using specific primers for the insert... so how can it be the result of lac-z ? also why transformation of correct plasmid results in both correct and wrong plasmid? the size of wrong size is allways the same

I used also 2 different Ecoli Strains. in the case of top-10 strain, all colonies were wrong in size but when using DH5-alpha strain, I get some correct and some wrong sizes. But in the wrong size also I dont lose the restriction sites and I confirmed a 1000 bp long fragment which is inserted into my insert (in wrong cases) using specific primers for the insert... so how can it be the result of lac-z ? also why transformation of correct plasmid results in both correct and wrong plasmid? the size of wrong size is allways the same