HELP! Tomato genomic library

Leonard N. Bloksberg bloksber at pilot.msu.edu
Fri Feb 11 14:28:00 EST 1994


In Article <1994Feb11.091215.897 at cathy.ijs.si> "Kristina.Gruden at ijs.si (Kristina Gruden, IJS)" says:
> Hi!
> 
> I have problems with preparing tomato genomic library. We're using Stratagene
> system and it works fine with human DNA. So I suppose that the problem is
> in the DNA contaminants. I'm using the folowing protocol for DNA isolation:
> 1. isolation of tomato nuclei (Honda buffer)
> 2. lysis of the nuclei 
> 3. some polysacharides are still present, so I use CTAB purification together 
>    with phenol-chloroform extraction
> 4. I precipitate DNA with 100% EtOH and wash it with 70% EtOH and Sodium acetate
> pH=5,2
> 
> The titer of the library is very low - only 10000 phages/ml.
> 
> Any suggestions are welcome
> 
> Kristina
> 
.
When I made my tomato library I started my DNA prep very similar to yours,
but I skipped the CTAB.  Instead I double CsCl banded the DNA.  This worked
very well.  I always had problems with the CTAB protocols with tomato for
some reason.  Good luck.
.
Leonard N. Bloksberg
bloksber at pilot.msu.edu
Dept. of Crop and Soil Science
Michigan State University
East Lansing, MI  48824
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