DNA Isolation Protocol

DELANEY 919-541-8577 DELANEYT at am.abru.cg.com
Tue Apr 4 13:32:06 EST 1995


Lynn Reuber asks if..

>anyone out there have a good protocol for making plant DNA 
>from 1-2 g of tissue without dealing with CsCl?  Has 
>anyone had experience with making DNA from seedlings? 

Lynn,  ..I tried to reply to you directly, but my mailer won't reply to your 
address, and you didn't provide your e-mail address, ...so at the risk of 
boring others, my reply is shown below.  

I'll take this chance to request that persons posting messages to the BB 
should include their e-mail address in the body of their message  ;-)

==================================================================

Lynne,
 
I have had good success with the procedure in Dellaporta et al, Plant Molec 
Biol Reporter 1: 19-21.  The protocol also works well when scaled down to an 
eppindorf-size prep on 15-500 mg tissue, such as for small seedlings (ie 
scale down reagents to 600 ul Ex Buf, 40 ul SDS, 200 ul KOAc, 400 ul 
isopropanol).  For RE digestion adding spermidine to 5 mM often makes a big 
difference (add AFTER combining DNA and RE buffer/salts to prevent 
spermidine pptn of DNA!).

Terry Delaney
Ciba Ag Biotech
delaneyt at am.abru.cg.com





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