Biomaterials Engineering, Day 3, 5/4/07
This shows a minimal effect of temperature on non-specific binding.
- To find the most efficient gold-binding yeast from our clonal cultures.
- Place a 6 mm x 10 mm slide into each well of a six well dish, shiny-side up.
- Add 2 ml Gal Blocking/Binding Buffer to cover the gold slides.
- Place the dish on the rocking shaker set at speed 7 and let it rock there for at least 10 minutes.
- The teaching faculty provided 5ml aliquots of each yeast control group. Pour the contents of the overnights into 10 mL falcon tubes. Harvest the cells by spinning the tubes in the centrifuge 2000 RPM, 5 minutes.
- Aspirate the media from the cells. You do not have to remove every drop. In fact it’s better to leave a small amount of liquid on the cells rather than risk aspirating away the cells themselves.
- Resuspend the cell pellet in 1 ml of the Gal Blocking/Binding Buffer
- Aspirate the Blocking/Binding Buffer from each of the gold slides and pipet 1 ml of fresh Gal Blocking/Binding Buffer.
- Add the cells that you have resuspended into the appropriate well.
- Place the dish on the rocking shaker set at speed 7 for 30 minutes.
- After panning for 30 minutes, move the gold slides to the bottom three wells of the six well dish with 2 ml fresh Gal Blocking/Binding Buffer. Use forceps to move the slides, touching only the edges, and wipe the forceps with ethanol between each sample.
- Place the dish on the rocking shaker set at speed 7 for 15 minutes.
- Label six Petri dishes that have –trp media. They should be labeled with the name of the sample as well as the date and your initials.
- Use forceps to move the slides one last time into a new six well dish with 2 ml fresh Gal Blocking/Binding Buffer.
- Photograph the surface of the gold slides using the digital camera and the WILD® light microscope.
- Move the gold slides to eppendorf tubes that have 500 μl of sterile water. Vortex each for 30 seconds exactly. Plate 100 μl on –trp Petri dishes. Once all your plates have dried, wrap them with your colored tape and place them in the 30°C incubator, media side up.
- You can aspirate any remaining liquid from the 6 well dishes and discard them into the biohaz waste. The eppendorf tubes with your yeast and gold slides can be directly discarded into the biohazardous waste.
- Negative Control
- pCT-CON plasmid will not bind gold
- Positive Control
- pAu1 plasmid known to bind gold
The optimization results showed no reason to modify our protocol between the first and second screen. We therefore repeated the same protocol and will look on Wednesday to find the best binding motif and send it for sequencing. We also set up a time this weekend to meet and get a good start on our presentation.