Troubleshooting

Start of the third week and back to performing CAT ELISAs. I am constantly reminded of how important getting the details right is to successful experimentation. Some of these details that I have failed to remember or perfect in past and current CAT ELISAs are: making sure the protease inhibitor tablets have completely dissolved in the lysis buffer before adding the lysis buffer to the plates, inverting the CAT Enzyme Working Dilution tube multiple times and pipetting the solution up and down to try to ensure a uniform distribution of CAT Enzyme within throughout the solution, making sure the cold centrifuge is set at 4˚ Celsius, checking the multi-channel pipette to ensure that each pipette tip has actually taken up solution, loading the sample trays in the CAT ELISA plate in the correct orientation so the plate reader will read them, and famously (from my first attempt at a CAT ELISA), not using working dilutions as washing buffers. These are only small parts of the CAT ELISA procedure, but remembering and performing them makes all the difference in the world.

On another note one of the post-transfection plates fluoresced green under the fluorescence scope today, showing that transfection with GFP-tagged Thyroid Hormone Receptor Alpha had occurred and transfection efficiency is one less source of error to worry about (for this round of experimentation at least). In addition to the successful transfection, when seeding plates on Friday and loading the hemocytometer to approximate the cell concentration, there were 69 cells in the central grid which correlates to 6.9 x105 cells per plate. The CAT ELISA protocol requires seeding at 6.0-7.0 x 105, so plate seeding is also less of a concern at the moment. The reason I mention a small and rudimentary part of the process like plate seeding is that it’s another detail that could hinder successful experimentation if performed incorrectly. Similarly, transfection seems like a simple process but carelessness is costly and adherence to the protocol is critical to achieving high rates of transfection efficiency. I’m still undecided about whether or not plasmid dilution is worthwhile, I’ll have to accumulate more data to come closer to a decision regarding future plasmid dilutions. Going forward, I hope that remembrance of the details and successful transfection will help achieve better results.