HELP! Tomato genomic library

Dr. C.P. Healy chealy at crc.ac.uk
Fri Feb 11 07:12:12 EST 1994


In article <1994Feb11.091215.897 at cathy.ijs.si> Kristina.Gruden at ijs.si (Kristina Gruden, IJS) writes:
>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.
>
[B[B>Any suggestions are welcome
>
>Kristina

A post-doc who I once worked with also had problems making a library from 
tomato genomic DNA. The reason was probably because of the large amount of
methylated DNA in the tomato genome (methylated cytosine in particular). 
Are you using mcr- host strains *and* packaging extracts. If not then I 
suggest that you take this precaution.
Good luck!




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