Plant DNA **********
jakku at mrna.tn.nic.in
Wed Sep 20 05:57:11 EST 2000
try Dellaporte et al procedure. It is easy and uses 1-7% PVP to remove the polyphenols problem. I have managed to obtain very good quality DNA even from oilseed crops like Sesmum and other polyphenolic rich plants like Bougainvillea.
best of luck
----- Original Message -----
From: Savita Shah
To: parvaizqazi at my-deja.com ; methods at hgmp.mrc.ac.uk
Sent: Wednesday, September 20, 2000 2:52 AM
Subject: Re: Plant DNA **********
Here is the protocol we follow routinely in our lab..
100mM Tris HCl pH7.5 or 8.0
20mM EDTA pH7.5 or 8
1% sodium meta bi sulfate
1%bMe (add fresh)
5mM thiourea (optional.. helps if you have lot of polyphenols that are difficult to get rid off)
1.. 1. Grind Tissue (0.3g) in liq. Nitrogen
2.. 2. Add 0.9ml 2X CTAB buffer (preheated at 65degC is better)
3.. 3. Incubate at 65degC waterbath for 15min. (better if you invert tubes once or twice while they are in waterbath)
4.. *4. Add 0.5-0.9ml chloroform:iAA (24:1) centrifuge @14krpm, 5min.
5.. * Sometimes there is not enough room for the chl:iAA in the same tube..in that case, centrifuge the tubes (14krpm, 5min.) after removing from 65 waterbath, collect supernatent in fresh tube and follow chl:iAA step)
6.. 5. Repeat step 4
7.. 6. Collect supernaten and add 0.6vol of 100% iso-propanol
8.. 7. Delicately invert tubes to ppt DNA..
9.. 8. You could either spool the DNA (if it is spoolable) or let the mixture stand at RT for 15 min.
10.. 9. Spin the DNA (1 min at 14krpm)
11.. 10. Wash DNA (once or twice) with 70% ethanol. Drain the ethanol. Again spin the tubes briefly and carefully pippete the residual ethanol.
12.. 11. air dry and dissolve DNA in 200microL TE(Tris 10mM, EDTA 1mM, pH 7.5 or 8)
With this protocol using maize leaf tissue.. I could get minimum 10ug DNA.. there is no problem to carry RE digestions, run Southerns or PCR.. works great
I like this protocol since you do not have to use any phenol. pHing phenol is tricky.. acidic phenol may fragment your DNA.. so if you are using phenol in your protocol.. please check pH of phenol:)
parvaizqazi at my-deja.com wrote:
I am working first time on plant DNA isolation.I got a lot of
difficulties for standardisation of a protocol for isolating the
same.Finally somehow I am able to isolate the DNA but the problem now
is that when I check the DNA in the agarose gel a portion of the DNA is
degraded.Although I repeated the isolation many times the problem is
there.I request a suggestion or better if any body provides me with a
better protocol so that I come out of this problem.Thanking in advance.
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