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Yeast:e.coli shuttle vector for making Chlamydomonas expression library

Shawn Burgess jensen_lab at qmail.bs.jhu.edu
Wed Jul 5 09:55:22 EST 1995


There are a series of well made and well characterized expression 
plasmids for yeast described in NAR vol 22 No. 25 p.5768 (1994).  These 
vectors have the same problem as others you have mentioned,  but I would 
suggest two ways of dealing with this.  The first if you are confident 
of the quality of the cDNA library is to ignore the lack of the ATG.  If 
you engineer an ATG in you are effectivly making only 1 in three clones 
useful.  If the library is a high quality one this same efficiency can 
be approached.  The second option is to insert a small double stranded 
oligo into an upstream cloning site which inserts the needed ATG.  We 
have done this to insert other useful short sequences into vectors.  the 
major trick is to have the necessary enzyme overlaps in the oligo but 
when ligated in does not regenerate the cloning site.  If 
unphosphorylated oligo is ligated in the presence of the enzyme used to 
cut the vector originally,  then background is significantly reduced and 
normally only one copy of the oligo is inserted.  Just make sure that 
the oligos are long enough to stay annealed.  This worked very well for 
us and only takes a couple of days.  





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