Shreffler:Reporter Construct with Retinoic Acid

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Reporter Construct with Retinoic Acid

05/15/209

Growing a T-Cell Line: Jurkat Cells

Jurkat Cells: Tower 6, Box 1 in liquid nitrogen (Lab 17-46), Box Labeled: T6B1, #61 - 1 x 10^7 (9|5 possible strain), dated 10/10/2006

- Obtain cells from liquid nitrogen, thaw them out in hot water bath until a small pellet of ice remains in the cryovial - With a transfer pipet, transfer cells into 50 ml tube and slowly add 10 mls of PBS to allow the DMSO to be diluted - Spin cells at 300g for 10 minutes - Make a medium containing RMPI and 10% Fetal Bovine Calf Serum - Use a 25 cm^3 cell culture flask instead of a larger flask (since there are not too many cells, keeping them together will better ensure their survival) - Resuspend the cells in 1 ml of the RPMI medium - Add 5 mls of the RMPI medium to the culture flask, then add the cells with a transfer pipet - Keep the flask upright with the lid slightly open (these cells are suspension cells, therefore they will not stick)

Prepare an Agar Gel Plate:

- From a pre-made Agar gel solution, take out a 100 mls (this should make 4 to 5 plates) - Bring the solution to a slight boil and add 50 uls of carbinicillin (an antibiotic) - Allow to cool and pour into each plate (covering the complete plate) - Let it sit for 5 to 6 hours at room temperature, then place at 4 degrees to completely dry overnight - Plates must be turned upside-down to ensure no moisture or condensation affects the gel

05/18/2009

Plasmid: E. Coli - Tx 3X RARE

Growing the Bacteria:

- Spread the plasmid across the gel - Incubate at 37.0 degrees overnight, to allow the bacteria to grow, expand, and colonize

Prepare LB (Luria Bertani) medium: - Dissolve 5 g of tryptone, 2.5 g of yeast extract, and 5 g of NaCl in 450 mls of distilled water - Place on stirring plate with stir bar and allow to mix, until everything dissolves - After completely mixing, add enough water to bring the volume up to 500 mls - Divide into two 150 ml flasks and store remaining in a sterile bottle - Autoclave the two flasks and sterile bottles (30 min)

Inoculation of Bacteria for Overnight Culture

(Inoculation is the process of introducing an antigen into the body, in order to stimulate the production of antibodies to produce immunity to an infectious disease.)

- Use a flame to keep the LB medium sterile - Put 3 mls of LB medium into a 15 ml tube - The concentration should be 50 ug/ul of carbinicillin, so add 1.5 ul of the antibiotic to the 3mls of medium - With a sterile toothpick and tweezers, pick a colony off the plate and place the toothpick into the 3 mls of medium - Place in shaker overnight (approximately 16 hours) at 37.0 degrees and at a speed of 240 rpms - After the culture has been placed in the shaker overnight, carefully remove - Add 75ul of carbinicillin to the two 150 ml flasks of LB medium previously prepared - To start the next overnight culture, we have to do a 1 to 500 dilution, so take 300 ul of bacteria culture ( 1st overnight culture) and add it to each of the 150 ml flasks - Place in shaker overnight (approximately 16 hours) at 37.0 degrees and at a speed of 240 rpms