Maximum plasmid insert size?
Phillip San Miquel
pmiguel at bilbo.bio.purdue.edu
Tue May 9 18:51:20 EST 1995
My boss, Jeff Bennetzen, subcloned a 21 kb chunk of an Erwinia stewartii
cosmid as an EcoRI fragment into puc19. It contained the carotenoid
operon and turned coli strains bright yellow -- even orange under the
right conditions. He deleted out a KpnI fragment to bring it down
to 14 kb. But I used to hop Tn1000's into it to map the critcal
regions. These Tn1000 mutants were 23 kb (counting the vector.)
They grew fine in HB101 and DH5alpha and number of other strains.
But I don't think many chunks of Eukaryotic DNA this large would be
stably maintained at puc19 copy number levels. But a couple of guys
in our lab have cloned 14kb stretches of maize and sorghum DNA into
puc-like vectors such as pBS and pGEM without any trouble.
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