PBR322 - maximizing yields

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Aug 27 10:16:41 EST 1992


> The postings about how to get great yields of pUC are all fine
> and good, but in most cases we get enough DNA from a pUC
> miniprep to keep us happy for a long time. 
> 
> Unfortunately, some things can only be done in pBR-based
> vectors and I have been plagued with totally miserable
> yields of one particular example of same for more than
> a month now.
> 
> What are the best tricks for increasing yields of pBR-based
> vectors?
> 
> Brian
> 
I have had to face similar circumstances where I had to use the 
pBR322 based plasmids.  I found the following protocols to work
very well with the HIV genomic clone DNAs.  

I pick my colonies first thing in  the morning and incubate at 32C
until evening when I am ready to leave (about 8-10 hrs incubation).
I take out about 0.8 ml out of about 3 ml and add glycerol (15%) to 
store the cells and to the rest I have added anywhere from 25 to 150
ug per ml of chloramphenicol and let it grow overnight.  
By the next morning the cells are usually ready and I have had reasonably
good yields.  
Good luck.
Raj   Kit (!) Shankarappa.
Univ of Pittsburgh.




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