-
Sterilize Seeds and Sow on Plates
I use Arabidopsis thaliana Col-0 seeds and bath them in a solution of 5% bleach and 0.1% Triton-X in order to get rid of any fungal or bacterial remnants that might harm my plants while growing on the plates. -
Transfer Seedlings to Soil
The seedlings are fragile - one needs to be careful, especially not to injure the root of the small plant. -
Prepare Solutions Required
The protocol I follow is published here: Protocolhttp://www.ncbi.nlm.nih.gov/pubmed/17585298 As you might remember from courses at school, plants have cell wall which for examples mammals do not have. The cell wall is a rigid but flexible structure that helps the plant to stand upright.
For my project, I would like to find out, where the protein I am interested in is located inside the plant cell. Because proteins are so small, they.. -
...continued
can not be seen by regular microscopes. But there is a possibility to add a fluorescent "tag" to a protein and this allows us to see where the protein is sitting in the cell. However to get these kind of tagged proteins into the cell, one needs to "transform" the plant cells this means, we provide it with DNA which the cell then uses to make tagged protein.
To be able to transform the cell, we need to get rid of its cell wall using enzymes. The "naked" cell that remains is called "protoplast"! -
Prepare Enzyme and PEG Solution, Perform Protoplast Isolation and Transformation
The protocol is still as mentioned and I prepare all the remaining solutions, that need to be very fresh.
The procedure from isolating protoplasts to transformation takes almost an entire day; we need to isolate the protoplasts from their cell wall, then wash them several times to remove debris material of cell walls and finally prepare them for transformation, and because protoplasts are so tiny and fragile, everything needs to be done very slowly and carefully. -
Period: to
Overnight Incubation for Expression of Transgene
-
Check Localization of the Tagged Protein e.g. at Confocal Microscope
After a night's incubation during which the cells are able to make tagged protein from the DNA they have been given, we can finally look at our protoplasts under the microscope. It looks like the protein I am interested in is sitting on the outer margin of the cell, the plasma membrane.
