HELP! Ligation of tomato genomic DNA

[Robert Kuhelj, IJS]

Hi!

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
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.