HELP! Ligation of tomato genomic DNA

Bill Melchior wmelchior at ntet.nctr.fda.gov
Wed Sep 1 09:54:26 EST 1993


In article <1993Aug30.094353.530 at cathy.ijs.si>, 
Robert.Kuhelj at ijs.si (Robert Kuhelj, IJS) writes:
> 
> 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 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-

I have no experience with plant DNA, but I find precipitation from ammonium
acetate frequently improves the quality of "dirty" DNA preps from other
sources; i.e. A260/A280 ratios much improved.  (ref: Crouse & Amorese, BRL
Focus, vol 9 #2, p 3 (1987); and see the BRL catalogue (p R-34 of the 93-94 
edition.) 

Briefly:

1)  To the dissolved DNA in a low-salt buffer, add 1/2 volume 7.5 M
ammonium acetate, and mix. 

2)  Incubate 30 minutes.

3)  Centrifuge 15 minutes at 16,000 x g. (I frequently see a precipitate at 
this point, which is NOT DNA.)

4)  Add 2-2.5 x the TOTAL volume of 95% ethanol (I use 2x, which seems to
work fine, and I figure it's less likely to precipitate contaminants that
weren't removed in the previous step.), mix, and incubate at least 30
minutes, preferably overnight.  (I usually incubate an hour or more, and
get good recoveries as judged by gels.  Crouse and Amorese got marginally
better recoveries after overnight incubation than after 30 minutes.) (Note:
Use a large enough container.  The final volume will be 4.5 x the original
DNA volume if you use 2 volumes of ethanol.) 

5)  The BRL procedure doesn't call for it, but I rinse the pellet a couple 
of times with room temperature 70% ethanol to remove excess salt.

-Bill
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