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help:failure of extraction of plasmid DNA - (Aug/26/2005 )

I have ligate a 80bp linker with cohesive ends produced by digestion into a vector containing Amp resistance. I transform DH5a competement cells and pick the colonies( the negtive control has no colonies).

The problem is that I failed to extract plasmid using GFX microplasmid prep kit(Amersham). After purification, no plasmid band is shown in the agrose gel.

Since I can manage to get colonies, why did I fail to get plasmid DNA?

Thanks a lot for your suggestion.


Though am not very sure one of the possibility could be your plasmid could've got recombinated as DH5aplha is not rec resistant. Trry using better strains like XL1Blue or Stbl2. Also try normal miniprep for your same positive cells (if you have streaked plate).


The amp in your plates might be off. Happens all the time once the plates get old.


DNA sequencing

-Daniel Tillett-