isolation of DNA from bracken

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Mon May 2 18:24:01 EST 1994


>Hi
>
>   I am isolating DNA from bracken (pteridium aquilinum) using a CTAB 
> method. This method is fine for isolating DNA from a huge range of 
>plants. I can get bracken DNA to ppt in ethanol but after washing it, it 
>is almost impossible to desolve it all even when the solution is 
>warmed to 60 C for a few mins. The resulting solution is thick, similar 
>to fragmented salmon sperm,  with strands of undesolved DNA.  Spec 
>gives a value of 0.64 mg/ml.
>   The DNA is also difficult to cut with restriction enzyme ( this is not 
>due to the CTAB) and when run out on gel ethidium Bromide heavily 
>stains something in the well.
>
>Questions
>1. Does any one have a simple alternative method?
>2. Does someone out there know why my DNA fails to desolve? Could 
>it be co-purification of something with the DNA , if so how do I get rid 
>of it.
>
>Anthony Hall 
>ajwh1 at leicester.ac.uk

Hi Anthony

I have NO experience with plant DNA but genomic high molecular weight DNA
from higher organisms is very difficult to dissolve if it is not already
sheared.  With mammalian stuff (mouse, rat, human, cattle, pig, horse etc)
we routinely dilute to about 0.2mg/ml otherwise it comes out of solution at
4*C.  Even then I place it at 65*C for at least 30 min before trying to
pippette it (or it glugs)(overnight at 65 does not hurt if you dilute in
TE).  A very bad trick for young players is to use a speedy vac to try the
DNA after ethanol pptation.  The DNA then is EXTREMELY difficult to
redissolve.  I only dry under vacuum in a dessicator for 5-10 minutes. 
Even after you dilute, reheat at 65*C and re-read the OD's (they could be
all over the shop due to gluggy pippetting).

For restriction of this sort of DNA I use about 5-10u/ug DNA and don't need
to cut for more than an hour.

Good Luck, Klaus
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
Snail mail: Canberra, ACT 0200, Australia
E-mail: Klaus.Matthaei at anu.edu.au
Landline: 61 6 249 3782

"Isn't it funny.  
Some people NEVER have the time time to do things right.
But ALWAYS have time to repeat it?"
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