ligation of tomato dna

john hachey HACHEY at ABRSLE.AGR.CA
Thu Sep 2 14:08:08 EST 1993


>I'm trying to prepare a tomato genomic bank.The problem I have is that the DNA 
>I have isolated is not able to ligate. The procedure I've used for DNA isolation
>is:
>1. homogenisation of leaves in liquid nitrogen
>2. washing of homogenisate with methanol, 0.1% mercaptoethanol
>3. extraction of DNA in buffer containg 100mM Tris-HCl (pH 8), 100mM EDTA
>   1% PVP, RN-ase ( 20ug/ml), 1% SDS, proteinase K, incubation: 3h, 55C
>4. phenolisation
>5. CTAB precepitation of polysaharides: I followed the procedure described in 
>   Plant Molecular Biology Manual (1989)
>
>The DNA precipitated nicely. But it was dark coloured, the A260:A280 was
                              *************************   

You might want to add some ascorbic acid (~0.1%) to your exrraction buffer
to inhibit  the oxidation (of phenolics?) that results in the brown pellet

>only 1.2 and A260:A230= 0.5. So I dialised the solution against 10mM EDTA, 
>20 mM Tris-HCl pH=8 but the ratios haven't change.
>The DNA was normaly restricted With Sau3AI and EcoRI but didn't ligate.
>
>So if you have experience with plant genomic DNA or any other suggestions for 
>my problem please send me a message.
>
>			Kristina 
>
>
>Kristina Gruden
>Institute Jozef Stefan
>Department for Biochemistry and Molecular Biology
>University Of Ljubljana 
>Slovenia
>
>Replies to robert.kuhelj at ijs.si, please.
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