(no subject)

Lindsay Collinson p8620253 at acsusun.acsu.unsw.edu.au
Mon Feb 19 23:44:09 EST 1996


HELP!! pcDNAI plasmid deletions/instability

Is there any one out there who has had experience with the Invitrogen 
expression vectors pcDNAI or pcDNAI/AMP and/or experience with 
expression cloning using COS1 cells.

I am using these vectors to construct cDNA libraries and to transform 
COS1 cells for use in expression cloning.

Analysis of plasmid DNA isolated following several rounds of 
transfection, selection has revealed the presence of what appears to be 
'deletion' plasmids which were confirmed to be of pcDNAI vector origin 
by sequencing (appox. 2 to 3 kb in size compared to the full length 
4.8kb vector). They contain the necessary genes for survival during 
transformation into COS1 cells and antibiotic selection in E. coli.

I have used two libraries [one in pcDNAI (prepared by invitrogen) and 
the other in pcDNAI/AMP (prepared in-house] and both are producing 
similar deletion clones (the pcDNAI library in particular).

I have a feeling that repeated transformation of selected clones into 
COS1 cells induces some sort of plasmid recombination/instability 
resulting in plasmid deletion.

We transform COS1 cells using the Life Technologies transfection reagent 
'Transfectamine' and cycle clones into E. coli DH10B cells (also from 
Life Technologies.

Has anyone experienced similar problems with these vectors or has had 
experience with expression cloning using COS1 cells? 

I welcome your opinions.

Lindsay Collinson





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