RobWardenWNB:M4D1

=Biomaterials Engineering, Day 1, 4/27/07=

Selecting For Gold

 * Purpose: To select for sequences with a gold affinity in a library of yeast plasmids.

Protocol
 * 1) Place a 6 mm x 10 mm slide into the top three wells of a six well dish, shiny-side up.
 * 2) Add 2 ml Gal Blocking/Binding Buffer to cover the gold slides.
 * 3) Place the dish on the rocking shaker set at speed 7 and let it rock there for at least 10 minutes.
 * 4) The teaching faculty provided 5ml aliquots of each yeast group. Harvest the cells by spinning the tubes in the centrifuge 2000 RPM, 5 minutes.
 * 5) 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.
 * 6) Resuspend the cell pellet in 1 ml of the Gal Blocking/Binding Buffer
 * 7) Aspirate the Blocking/Binding Buffer from each of the gold slides and pipet 1 ml of fresh Gal Blocking/Binding Buffer.
 * 8) Add the cells that you have resuspended into the appropriate well.
 * 9) Place the dish on the rocking shaker set at speed 7 for 30 minutes.
 * 10) 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.
 * 11) Place the dish on the rocking shaker set at speed 7 for 15 minutes.
 * 12) 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.
 * 13) Use forceps to move the slides one last time into a new six well dish with 2 ml fresh Gal Blocking/Binding Buffer.
 * 14) Photograph the surface of the gold slides using the digital camera and the WILD® light microscope.
 * 15) 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.
 * 16) 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

Summary
Today we screened a library for sequences that will bind gold. We will narrow them down even more later, then sequence the plasmids to to determine a gold-binding motif.