Introduction
EGFP will be measured by direct visualisation with a UV microscope and the appropriate filters. By using an inverted microscope, we are able to assay the cells directly whilst they are still alive. The use of GFP (and its variants) has been a very powerful addition to the molecular biologist in recent years as real time experiments can be performed.
We will measure the percentage of cells expressing EGFP by physically counting several fields of view on the microscope. Although this is maybe not the most accurate method, it gives some idea of the transfection efficiency.
Aims
You will observe the HEK293/COS cells that you transfected on Monday with plasmids encoding EGFP and the P2X2-EGFP fusion protein. Record the transfection efficiency as judged by the % cells expressing EGFP, and the relative fluorescent intensity. Note, in all cases, the total amount of DNA used for the transfection is 2.0 µg.
EGFP
Materials required
Nescofilm Inverted UV microscope with EGFP filter
| Caution This practical involves the use of category II genetically modified organism-please follow instructions by demonstrators for safe handling and disposal. Take special care not to spill the cells Ð seal the plastic dishes with nescofilm. |
Record the % of fluorescent green cells and the relative fluorescence intensity of your groups′s transfection. The results of all the transfections will be discussed when all the data has been compiled.
Results
Discussion
Althougth data interpretation is somewhat subjective, this practial should demonstrate the relative efficiency of the three reagents.
Appendix
Filters/Fluorescent proteins
Clontech have developed a number of different fluorescent proteins that are excited and fluoresce at different wavelengths. Several companies specialise in filters for detecting use with GFP, such as GlenSpectra/Omega Optical (www.isa-gs.co.uk).
