Help on Cosmid minipreps

Ramanujam Raman ramjam at scripps.edu
Thu Oct 9 01:39:33 EST 1997


Hi Everyone,

I am looking for helpful suggestions on making miniprep DNA from cosmid
clones.  I have putative clones from an arabidopsis cosmid library.  the
library was package using Stratagene's Gigapack XL kit.  The DNA for the
library is prepared from BglII partially digested DNA ligated into
dephosphorylated BamHI cosmid vector.  I had several colonies after
transfecting the packaged DNA into vcs257 host cells (also from
Stratagene). 	 I picked some of these clones, inoculated 5ml O?N
cultures with Kan 25 ug/ml.  After 18-24 hours, spun down the cells and
did a basic alkaline-lysis miniprep.  My miniprep protocol is as
follows:

1) Resuspend cells in 150 ul of soln I (50 mM Glucose, 25 mM Tris 8.0,
10 mM EDTA) either vortexing or pipeting up and down.
2) Added 200 ul of 0.2N NaoH and 1% SDS. Mixed gently by inverting and
kept at RT for 5 min.
3) Added 150 ul of 3M KoAc. Mixed by inverting the tubes and kept on ice
for 5 Min.  
4) Centrifuged at 4C for 10 min 
5) Phenol-chloroform extracted the supernatant and ethanol precipiated
6) dissolved in TE (8.0) and ran on 0.6% gel

Now the problem I have been having is big smear of DNA all the way from
the wells of the gel.  It looks like I am pulling down genomic DNA more
than any amount of cosmid DNA.  I have also tried the qiagen column
miniprep method with the same result.  Phenol-chloroform extraction does
not seem to make any difference in the quality of the yield. 

Are genomic DNA smears commonly encountered while working with cosmids? 
Is there any efficient way of increasing the cosmid DNA yield?  I would
appreciate if anyone who has worked with cosmids suggests better
miniprep methods that overcomes the above problem.  Please send your
replies to my email address.  Thanks in advance for your time,

RAM. 

--------------------------------------------------------------
RAMANUJAM RAMAN 	       |	
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