A simple question
nurwin at csu.edu.au
Wed Nov 9 17:37:43 EST 2005
Have done plant genome sizing with PI and have used the Otto buffers using different plants with variable success. The plant you are using and it's developmental stage can greatly affect results. I would try several different buffers and technique and there are many variations. If your plant releases phenolics on chopping you need to include DTT or some other antioxidant/ reducing agent. So far I have found the following technique (which uses a modified Galbraith's buffer) in which your tissue is chopped and stained at the same time is OK for several species. For simple ploidy analysis (diploid/ tetraploid etc) use DAPI as it is a better dye (CV's) but you need a UV source for this. PI/RNase is OK and essential if you want to determine relative genome size. PI is not AT specific like DAPI. Hope this helps
Method 1 for DNA analysis of plant nuclei.
Galbraith's buffer (G) Buffer
This method I modified from one the University of Leicester Website
30mM trisodium citrate
0.1% triton X-100
pH 7.2-7.4 with 1M KOH (not less than pH 7.0 as DNases work well at <7.0.
Propidium iodide stock (PI)
Dissolve PI (Sigma 4470) in deionised water at 1 mg/ml Store at 4oC in dark bottle.
Different RNases can be used.
Used Sigma 5503 RNase A at 10 mg/ml in 10 mM Tris pH 8.0. Heat 95-100oC for 20 min to remove DNase activity. Aliquot and store at -20oC.
Dithiothreitol (DTT) stock
Dissolve DTT at 100 mM in deionised water. Stock in aliquots at -20oC.
Just prior to analysis mix the following on ice:
10ml of g buffer
500 µl of PI
100 µl of RNase
500 µl of DTT (optional to reduce effect of oxidative effects of releasing phenolics (browning))
1. Chop a small piece of tissue 0.5-1cm2 in a petri-dish or weighing boat in 1 ml of buffer. Filter through 30 um mesh or filter. Use fine razor blade and one blade per two samples only.
2. Place in flow cytometer.
3. If insufficient nuclei then chop more tissue to obtain best results. Pellet nuclei at 200g for 5 min in bench top swing out rotor. Resuspend pellet in 0.5ml of buffer the analyse in flow cytometer. You can also filter through a little plug of cotton wool before analysis.
Our instrument has a 488nm laser and I use a 570 LP filter on the photomultiplier. To obtain the best CV's the instrument must be aligned properly.
Dr. Nigel Urwin
Lecturer in Plant Sciences
School of Agriculture and Veterinary Sciences
Charles Sturt University
email: nurwin at csu.edu.au
From: jtigges at caregroup.harvard.edu [mailto:jtigges at caregroup.harvard.edu]
Sent: Wednesday, 9 November 2005 6:47 AM
Subject: RE: A simple question
I was wondering if anyone had any insight on staining plant nuclei with PI. We have a researcher who is staining plant nuclei using Otto I and then Otto II buffer with seperate PI(2 step procedure). We have been having problems establishing the DNA content model.
Thanks in Advance,
Core Facility Manager
Beth Israel Deaconess Medical Center
330 Brookline Avenue
Boston, MA 02215
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