Lab Protocols

Electrophysiology Chemicals 9/22/2004 Colin TTX                 from Alomone Labs, Israel. TTX w/ citrate. Na channel toxin 1 mg per ampoule, MW = 319.28, soluble in dH2O lyophilised = 1 year @ 4 deg C   /   liquid = 6 months @ -20 deg C desired final concentration = 1 uM 1 uM in 100 ml external solution = 0.031928 mg in 100 ml                         1 mg TTX in 1566 ul of dH2O => 50 ul aliquots each of 0.031928 mg                         aliquot solution is concentration of 2 mM      Picrotoxin         from Tocris Cookson. GABA A R channel toxin 1 g per ampoule, MW = 602.00, soluble in DMSO lyophilised = 1 year @ ...

Electroporator settings: For Xenopus: Brain or eye electroporation: Make sure to use capacitors   and set output to MONO                   DNA moves toward positive pole.                   DURATION                        1.6ms                   VOLTAGE                           30-70V (higher settings may kill animals)   Single cell electroporation/iontophoresis of dextans or DiI:                   Do not use ...

Reagents Below is a list of  catalog numbers for reagents we have commonly used: (product, company, catalog #) -DMEM (without glutamine), VWR, 45000316 -L-glutamine, Biowhittaker, 17-605E -Penicillin Streptomycin solution, Biowhittaker, 17-603e -Trypsin/Versene (EDTA), Biowhittaker, 17-161E -Phosphate Buffered Saline (PBS), VWR, 45000-434 -Iron-supplemented Bovine Calf Serum  (BCS), Sigma, C-8056   (or we have also used VWR  82002-978) -G418 sulfate, Calbiochem, 345810               For plates, we usually use VWR cat# 25382-166. We have never really noticed any difference in quality when we have ordered media components from different companies. However, several people in our lab feel that for tissue culture plates, Falcon (BD) brand is better than Corning. Alternative sources for these reagents:               Recently, we have been ord...

Materials • Complete medium: DMEM(high glucose) supplemented w/ 10% BCS and Gln (Pen/Streptmycin: optional) • Transfection reagents: i) 2M CaCl2: 2M in H2O, filter sterile, store at 4°C ii) 2xHBS: 8.0g NaCl, 0.37g KCl, 201mg Na2HPO4•7H2O, (1.0g glucose), 5.0g HEPES/500ml (adjust pH to 7.05 with NaOH and filter sterile, store at 4°C) • DNA • Chloroquine stock, 25 mM in PBS (freeze in vials) • (Optimem supplemented w/ ITS: optional) Procedure The following protocol is for 10 cm dish (medium: 10ml). If you use 6-well or 12-well plates, total volume of the medium should be 3ml and 1.5ml, respectively, and decrease the amount of each reagent accordingly. 1. Plate cells the night before to give 60-80% confluence at the day of transfection. 2. One hour prior to the transfection, change to medium containing 25µM chloroquine (from x1000 stock in PBS, stored at -20°C). Volume should be 10 ml per dish. (Chloroquine can be omitted, but increases efficiency about x2) ...

Coverslip preparation: Clean up coverslips: Immerse coverslips in 100% EtOH for 20 min Rinse in d.d. H2O for 3 times Imerse coverslip in d.d. H2O for overnight Autoclave Dry Poly-L-Lysine solution: Filter-sterilize 0.1M broic acid (pH=8.3 w/ NaOH) Dilute Poly-L-Lysine (50X) in boric acid to the final concentration of 50 μg/ml Coat coverslips: Lay out coverslips in small Petri-dishs Place 50 μl Poly-L-Lysine in the center of the coverslip Push down on coverslip with pipette tip as you add solution to make sure it is flat Let air dry for 1 hr Rinse the coverslip with sterilized H2O Air dry These coverslips can be stored at room temperature for more than one month. Tectal cell culture 1.        Anesthetized tadpoles in Filter-sterilized 0.1% MS222 Dissect brains in sterile HBST and collect them on ice Transfer brains into sterile Ca/Mg free HBST+EDTA w/ fire-polished Pasteur pipette Cut tecta into small pieces (100-200 um) Rinse these pieces wit...

Hahn Lab - Methods - EMS Yeast Mutagenesis EMS Mutagenesis of Yeast Linda Hoskins, Hahn Lab Last modified10/16/98 1. Grow a 50 ml O/N YPD yeast culture at 30 degrees C. 2. Determine cell density and transfer an amount of culture corresponding to 1 x 108 cells to several 15 ml conical tubes. Spin down cells and wash with 5 ml sterile water. Spin down cells and wash with 5 ml 0.1M sodium phosphate (NaH2PO4) buffer, pH 7.0. Spin down cells and resuspend in 1.7 ml buffer. 3. Transfer cells to glass culture tubes. In hood, add 50 ul EMS (ethyl methanesulfonate) (also have one control tube without EMS). Incubate on roller at 30 degrees for varying amounts of time, between 20 min. and one hour. 4. At each time point, add 8 ml sterile 5% sodium thiosulfate (autoclaved). This will stop the mutagenesis by inactivating the EMS. Each cell suspension should contain 1 x 107 cells/ml. Save an aliquot of cells at this point to use in figuring out the cell survival. * All glassware, plas...

Hahn Lab - Methods - Yeast Sporulation Sporulation and Dissection of Yeast a/alpha Diploids Linda Hoskins Last Modified Tue, Nov 6, 2001 These conditions work well for the Hieter yeast strains 1. Isolate diploid colonies. Inoculate 2 ml YPD + Ade with a single, large diploid colony and grow O/N at 30 degrees C to stationary phase. 2. Put 200 ul of stationary phase cells into 5 ml sterile, distilled H2O. Spin down 2 min. in clinical centrifuge. Wash again with 5 ml H2O. 3. Resuspend in 2 ml 0.5% (50 mM) KOAc (pH 7.0) + 0.5X nutrients for auxotrophic markers (usually Ade, Ura, Trp, Leu, His, Lys, Met). 4. Incubate on tube roller at rm. temp. for 3-7 days. 5. Check for sporulation under microscope. Spin down tetrads in a small glass tube and wash 3 times with 5 ml sterile H2O. Resuspend in 2 ml H2O. 6. Spin down 180 ul cells. Resuspend in 90 ul Zymolyase, 0.5 mg/ml in 1M sorbitol. Incubate at 30 degrees for 5-10 minutes. 7. Slowly add 0.3 ml H2O on ice to stop the rea...