pcDNA 1.1 Amp rearrangements

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Sun Sep 21 13:34:19 EST 1997


Good day Popes of Molecular Biology --
	I have been trying to clone various genes around the lab into 
InVitrogen's pcDNA 1.1 Amp, and I regularly find that a fair number of 
colonies look like they have my insert (upon digestion), but that the 
vector is the wrong size.  The vector should be 4.8 kb when linearised 
(and in my control lane the linearised vector runs at 4.8 kb), but in 
these strange transformants the cut out vector invariably has a 
molecular weight of ~3.5 kb.  I believe that band is the vector because 
there are no bigger bands (eg anything at 4.8 kb), and the other bands 
will correspond to what I would expect for my insert.  I do not think my 
enzymes are cutting the vector anywhere besides the multiple cloning 
region (the enzymes are listed by the manufacturer as unique cutters).  
The irritating thing is that in my hands pcDNA 1.1 Amp behaves as a 
medium copy # plasmid, whereas this putative deletion product acts as a 
high copy # plasmid.
	Has anyone seen this before or know what it is?
	Thanks much,
		Harry



More information about the Methods mailing list