A simple question

Urwin, Nigel nurwin at csu.edu.au
Wed Nov 9 17:37:43 EST 2005


Hi John

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 

 

G buffer

45mM MgCl2

30mM trisodium citrate 

20mM MOPS

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.

 

RNase stock

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.

Regards

Nigel

 

Dr. Nigel Urwin

Lecturer in Plant Sciences

School of Agriculture and Veterinary Sciences

Charles Sturt University

Wagga Wagga

NSW 2678

 

ph: +61-269-332450

fax:+61-269-332812

email: nurwin at csu.edu.au

-----Original Message-----
From: jtigges at caregroup.harvard.edu [mailto:jtigges at caregroup.harvard.edu] 
Sent: Wednesday, 9 November 2005 6:47 AM
To: cyto-inbox
Subject: RE: A simple question

 

Hello All,

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,

 

John Tigges 

 

Core Facility Manager

Flow Cytometry

Beth Israel Deaconess Medical Center

330 Brookline Avenue

Boston, MA 02215

(617) 667-4901

(617) 975-5536

 

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