Bryan Hernandez/20.109/Lab notebook/Module 4/Day 1

--Bryanh 16:02, 27 April 2007 (EDT)

purpose: familiarize ourselves with the protocol for which we will select for peptide sequences that bind to a specific material (of our choosing). We are using a library based approach.

Protocol
Some of the work for this experiment has already been done for you. Two nights ago, the yeast strains needed for today were grown to saturation in glucose-containing media at 30&deg;C. A lack of tryptophan in that media insures that all the yeast carry the pCT-CON, pAu1 or library plasmids. Yesterday, an aliquot of the cells was moved to fresh media (“subcultured”). This time galactose containing media was used to induce display of the fusions. The cells on your bench have been grown at room temperature for at least 20 hours.

Panning for Gold
1. Prepare the gold slides for study by incubating them in Blocking/Binding Buffer. Wear gloves when you handle the slides and touch only the edges. Place a 6 mm x 10 mm slide into the top three wells 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. 2. While the gold slides are blocking, move 5 ml of each strain to a 15 ml falcon tube. Be sure to swirl and fully resuspend the cultures before removing any cells. 3. Harvest the cells by spinning the tubes in the centrifuge 2000 RPM, 5 minutes. 4. 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. 5. Resuspend the cell pellet in 1 ml of the Gal Blocking/Binding Buffer 6. Aspirate the Blocking/Binding Buffer from each of the gold slides and pipet 1 ml of fresh Gal Blocking/Binding Buffer. 7. Add the cells that you have resuspended into the appropriate well. 8. Place the dish on the rocking shaker set at speed 7 for 30 minutes. During this time you should familiarize yourself with screens and libraries. You might begin by looking at for starters. 9. 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. 10. Place the dish on the rocking shaker set at speed 7 for 15 minutes. 11. Label three Petri dishes that have –trp media. They should be labeled with the name of the sample as well as the date and your initials. 12. Use forceps to move the slides one last time into a new six well dish with 2 ml fresh Gal Blocking/Binding Buffer. 13. Photograph the surface of the gold slides using the digital camera and the WILD® light microscope. 14. Move the gold slides to eppendorf tubes that have 500 &mu;l of sterile water. Vortex each for 30 seconds exactly. Plate 100 &mu;l on –trp Petri dishes. Once all your plates have dried, wrap them with your colored tape and place them in the 30&deg;C incubator, media side up. 15. 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.

resutls


As you can see the negative control has at least a few colonies, either due to a systematic error, which is extremely unlikely given the simplicity of the protocol. The other explanation is that our rinsing was not sufficient to remove all of the yeast that might be sticking to the slide through some small interaction. Improper blocking would also cause this phenomonon, however it's really hard to mess that up, so it is unlikely that this is the reason as well. The positive control was as expected, with tons of yeast. The experimental did have yeast on the slide at about the same frequency as the negative control and so it is hard to say if we actually found any gold binders. More tests would need to be done to verify this.