Lab Protocols

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...