Help on Cosmid minipreps

Koen De Smet k.desmet at nospam.ic.ac.uk
Fri Oct 10 07:50:17 EST 1997


Ramanujam Raman wrote:
> 
> 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,
> 
Dear Ram,

I am not sure where you are going wrong, but can give you my method for cosmid preps 
(below).
I used to do loads of them around 5 years ago, and they always worked. It also avoids 
the use of phenol, but the prep still contains RNA.  I used to do the digests on this 
cosmid/RNA mix and then add loading buffer with RNAse, leave that for 10 minutes and 
laod on a gel.

METHOD
Do as you normally do up to step 4, but using 100, 100 and 75 ul at each step.
After centrifugation, take 250 ul sup and add 500 ul ethanol.
Leave 20 minutes at -20°C.
Spin 5 minutes..
Dissolve pellet in 100 ul 0.3 M NaAc, pH 7.0; 1 mM EDTA.
Leave 15 minutes at R/T.
Add 200 ul ethanol.
Leave 20 minutes at -20°C.
Spin 5 minutes.
Dissolve pellet in 25 ul TE.


Koen De Smet
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