slblowe at HAPPY.UCCS.EDU slblowe at HAPPY.UCCS.EDU
Thu Aug 26 16:10:29 EST 1993

Dear Networkers,
	Has anyone had difficulty isolating inserts from Stratagene
lambda libraries? I though the problem of direct excision into bluescript
was solved with the introduction of SOLR cells. Because XL-1Blue cells
seemed to be slow (and sometimes inconsistent) growers, we contacted the
technical department of Stratagene. They said DH5a cells would be ok to use.
Since using DH5a cells, we have not been able to get an insert by either
a plate lysate or the direct excsion procedure (after going through 3 rounds
of plaque purification). They seem unstable.

1. Does anyone have experience with this? We are not sure if our problems 
are because of the DH5a cells, suggesting that we need to go back to the
original plugs and repurify them using XL-1Blues (which were used originally).
Even if there is a problem with the direct excision method, the plate
lysate method is usually reliable-it has routinely been used to isolate clones
from mammalian libraries. Or, maybe our yield of DNA is not high enough to
see the insert on a gel (although the lambda is there). Is the yield of
DNA dependent on the size of the plaques when the lysate is made? I would
appreciate comments and a successful procedure for either method.

2. Has anyone tried C600 cells? These are routinely used for mammalian
libraries. Does anyone have a source (free) of them?

3. Has anyone used lambda oligos to pcr out an insert from a plaque?
Would this be a reliable method, and if so, where can I get the oligos
and what size are the oligos.

We would appreciate any comments. I will summarize the answers for the
network. Thanks

Sandy Berry-lowe
slblowe at uccs.edu

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