subcloning large fragments

[brett]

>When subcloning DNA fragments larger than 5 kb into pBluescript, I 
>frequently recover DH5a mini-prep clones with various-sized deletions or 
>no inserts. What reagents (bacterial strain, plasmid vector, restriction 
>sites) should I be using so that I can subclone large (5-15 kb) DNA 
>fragments that are intact?

We maintain RNA viral genomes as cDNAs (10-13kB) in low copy plasmids with
fairly good luck. Try pACYC, or pBR322, or derivatives thereof. Bluescript
is pretty high copy #, so this might help. Also, I tend to use MC1061 for
routine manipulations but have resorted to WM1100 or SURE for things that
like to recombine.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu