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[Yeast] 2 hybrid library amplification and plasmid isolation problems

Gregory Finnigan gfinnigan at gmail.com
Wed Aug 17 00:04:45 EST 2005


I am a new graduate student rotating in a yeast lab and I am having
trouble amplifying the James genomic library (C1,2,&3) for a 2-hybrid
screening using GAL4 as the construct.  I have very old samples of the
plasmid DNA as well as new libraries in E. coli.  I have tried
amplifying the libraries (from the old plasmid DNA) using TOP10
electrocompetent E. coli and cannot get a high enough transformation
efficiency.  Thus, with new libraries (in E. coli) I am trying to
fomulate a protocol for amplifying the 5 x 10^6 clones properly and
isolating the plasmids (at least 100-500 ug DNA).  Thus far, using the
QIAGEN HiSpeed Midi-Prep Kit I have only been able to extract 5 ng/ul
plasmid.  Is there any protocol for amplification as well as
hints/suggestions for getting higher yields on large-scale plasmid
preps?  Thanks in advance for any help!

G



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