Preparation of the Arabidopsis protoplast
Place:Biology Experiment Teaching Center of Sun Yat-sen University
Experimental apparatus:Shaking table, 50 ml centrifuge tube, filtrating equipment, analytical balance, measuring cylinder
Experimental drugs:Cellulase hydrolysate, W5 solution
Procedure:
-
the configuration of Cellulase hydrolysate
Cellulase 0.225g Mecerozyme R10 0.045g mannitol 1.09g KCl 1.5g MES 4.18g water 12ml cooled down to room temperature after waiting in 55℃ water 10min. CaCl2 1.11g BSA 1ml water 2ml filtrating - Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml Cellulase hydrolysate.
- Put them in shaking table for 30min in the dark, then waiting for another 3h without shaking. When it comes to green and then gently shake it.
- (Adding 5ml W5 solution and then filtrating.)
- The protoplast was observed under the microscope
Introspection:
- Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.
- The shaking table should not exceed 80r/min.
![image](https://static.igem.org/mediawiki/2016/a/a6/T--GDSYZX-United--project_prepration1.jpg)
Filtrate during the configuration of Cellulase hydrolysate
![image](https://static.igem.org/mediawiki/2016/d/da/T--GDSYZX-United--project_prepration2.jpg)
Cut out the leaves
![image](https://static.igem.org/mediawiki/2016/c/cf/T--GDSYZX-United--project_prepration3.jpg)
Immerse Arabidopsis leaves in 5ml Cellulase hydrolysate.
![image](https://static.igem.org/mediawiki/2016/e/e8/T--GDSYZX-United--project_prepration4.jpg)
Prepare the microscopic slides
![image](https://static.igem.org/mediawiki/2016/2/2f/T--GDSYZX-United--project_prepration5.jpg)
Cellulase hydrolysate under microscope (nothing).
![image](https://static.igem.org/mediawiki/2016/c/c8/T--GDSYZX-United--project_prepration6.jpg)
Plant cells under microscope.