Standard Operating Procedure GARNet 4
Preparing Arabidopsis thaliana plants for analysis.
Author: Jennie Lewis
Last Modified: July 2003
Standard Operating Procedure:
Preparing Arabidopsis thaliana plants for analysis.
Materials and Equipment:
Pestle and Mortar
Polystyrene box
Ice
Liquid Nitrogen
Glass vials
Spatular
Methanol
Acetone
Blue roll
Procedure:
For plants to be analysed as fresh material:
1.
Chill a pestle and mortar in a –80C freezer for 30 minutes.
2.
Fill a polystyrene box with ice and cover the ice with tin foil.
3.
Make a well in the ice and place the mortar in the well.
4.
Pour a little Liquid Nitrogen into the mortar and allow it to evaporate.
5.
Remove plant material from the –80C freezer.
6.
Batch plant material of the same ecotype and tray number (e.g.
Ecotype GRN 101 Tray 1) by emptying all centrifuge tubes containing
the specified material into the pre chilled mortar.
7.
Keep material not being ground at -80C until grinding can be carried
out.
8.
Pour liquid nitrogen into the mortar and use the pestle to grind the plant
material. Grinding should be continued until the material is fine and
homogenous. More liquid nitrogen should be added if it all evaporates,
leaving the mortar dry.
9.
Allow any excess liquid nitrogen to evaporate off.
10.
Label a glass vial with GRN Number, plant growth number, plant
growth stage, date and name of operator.
11.
Remove the lid of the the labelled vial and transfer the ground material
to the vial, with a spatular.
Page 1 of 2
Standard Operating Procedure GARNet 4
Preparing Arabidopsis thaliana plants for analysis.
Author: Jennie Lewis
Last Modified: July 2003
12.
Retain the material at –80C until analysis can be carried out.
13.
Record location of the stored material.
14.
Between grinding material of different ecotypes allow the pestle and
mortar to cool to room temperature. Clean the mortar by pouring
approximately 10ml of methanol into it, wipe it clean and dry with blue
roll then use the blue roll to wipe clean the pestle and spatular. Repeat
this process with acetone. Re chill both the pestle and mortar by
placing in a –80C freezer for 30 minutes.
For plants to be analysed as freeze-dried material:
1.
Place, centrifuge tubes containing plant material, taken straight from
the –80C freezer, in to a freeze-drier for 48 hours.
2.
When the plants are dry batch plant material of the same ecotype and
tray number (e.g. Ecotype GRN 101 Tray 1) by emptying all centrifuge
tubes containing the specified material into a mortar.
3.
Keep material not being ground at -80C until grinding can be carried
out.
4.
Use a pestle to grind the material until it resembles the consistency of
icing sugar.
5.
Empty the mortar onto a clean piece of paper with a fold down the
middle.
6.
Label a 1.5ml glass vial with GRN Number, plant growth number, plant
growth stage, date and name of operator.
7.
Remove the lid of the the labelled vial and transfer the ground material
directly from the paper to the vial.
8.
Store the vial at –80C in plant material storage boxes.
9.
Record loaction of stored material within boxes on stored plant material
box information sheets.
10.
Between grinding material of different ecotypes clean the mortar by
pouring approximately 10ml of methanol into it, wipe it clean and dry
with a piece of blue roll then use the blue roll to wipe clean the pestle.
Repeat this process with acetone.
Page 2 of 2