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transformation of a big plasmid in E. coli - something wrong is happening in my transformation (Dec/29/2005 )

Hi, everybody!

First of all, sorry my english...

I need to transform (by heat shock transformation) a big clone (14kb - where the plasmid is pBR322 and my insert has 9kb). By miniprep, I see that something enters in bacteria, but a big part of my insert is being deleted. By a digestion, I see that my vector is intact, however only 2kb of my insert is intact.

Could the bacteria (Stable) delete part of my insert????
And what can I do about it?????

Thank you so much for your attention.


So you mean your insert is 9kb and vetor ~5kb!! huh.gif Anyway try using recombinase minus strains such as XL1Blue or Stbl2 to avoid recombination after tran formation.


my boyfriend has corrected me...
it is "sorry ABOUT my english"

sorry, sorry and sorry again and ABOUT other mistakes!!!!


What are those other mistakes which your boyfriend corrected?


He said only about that mistake...

The others are possible mistakes!!!

I don´t write in english for many years... If I could write in portuguese... smile.gif

And thanks for your suggestion!!


And I forgot to say that I use STBL to transform...
So, I don´t know what can be...


As i can make it out..since the plasmid is too big probably the cells are throwing out/losing the plasmid.But even then holding only 2kb and throwing out the rest is not reasonable/logical.I would suggest you increase the antibiotic conc. that you're using to grow the cells...i guess it must be try increasing the Amp conc to say 200 microg/microl