HELP! Need to make ultra-complex plasmid library

Ben-Sapiens srutt at beagle.Colorado.EDU
Fri Aug 5 16:50:01 EST 1994


I need to produce 10(8) or more transformants of E.coli strain
JM107 from ligated plasmids. 

1) I plan to linearize a plasmid such that it has a 3' and a 5'
overhang on its ends and will ligate a randomized oligo to 
re-circularize the plasmid, then probably fill in the single
stranded region. Has anyone done this sort of thing before?
How can I maximize the number of the ligated plasmids before 
I transform?

2) I plan to electro-transform the JM107 cells with at least
a microgram of ligated plasmid in such a way that at least
10(8) of them receive the plasmid and survive before the 
selection. Has anyone been able to produce this many
transformants from ligated plasmid? Does electro-transforming
JM107 result in multicellular filaments like JM109? Has
anyone tried using pantoyl lactone? How can I maximize the
number of transformants?

Thanks in advance for any information, advice or references
you can give me.

steve ruttenberg   srutt at boulder.colorado.edu



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