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Wipe down inside of Tissue Culture hood with 70% EtOH 2. Ensure that all media is warmed to 37C before using 3. Before moving any items under hood, dry with a paper towel, spray down with 70% EtOH, and dry again 4. Spray down gloved hands with 70% EtOH before working under hood 5. All incubations, unless otherwise noted, occur in a humidified incubator at 37C, 5% CO2 6. Always note passage number on plates. 7. Change media at least every 72 hours. For 10-cm dishes: To change media: Aspirate media and add 12mL of complete media (One part Eagles Minimum Essential Media, one part F12 media, 10% FBS, 1% pen/strep). Return plate to incubator To split cells: 1. 2. 3. 4. Aspirate old media Add 2mL Trypsin-EDTA solution directly to cells, incubate @ 37C for 3 minutes Add 10mL complete media to cell/trypsin solution and pipette up and down to resuspend Distribute cells to new 10-cm dishes as needed, then add complete media to 12mL. a. For a typical 1:2 split, distribute 6mL cell stock each to two new plates, then add 6mL of complete media to each plate. 5. Return plate to incubator For TC Flasks: To split cells: 1. Aspirate old media 2. Add 2mL Trypsin-EDTA solution directly to cells, incubate @ 37C for 2 minutes 3. Add 8mL DMEM (+ appropriate antibiotics)to dissociated cells. Pipette up and down to resuspend 4. Distribute cells to a new flask as needed, then add complete media to 12mL: a. For a 1:20 split, add 18mL of media to a flask, and 500 µL of the cell stock To plate into 12-well plates: 1. From the cell stock generated above, mix 1mL of cell stock and 11mL of DMEM in a 15-mL falcon tube 2. Distribute 1mL of cells to each well of a 12-well plate 3. Cells should be ready to transfect the next day
Scrape and collect in microcentrifuge tubes.General Protocol for N2A Lysis and Western Blotting of Lysates Cell Lysis: 1. foam pad. 400µL β-mercaptoethanol. Cut whatman gel blot paper slightly smaller than foam pads from the transfer apparatus 5. Make up Tris-Glycine buffer by diluting 5x stock in milliQ H2O and completing to 1X with MeOH so that the final concentration of MeOH is 20%. 3. Aspirate media from each well of a 12-well plate 3. Cover plate and incubate 30 minutes at 32C Uncover plate and load into a 96-well plate reader. BCA Assay 1. +. keep tubes on ice. foam pad. Run gel at 180mA for 75 minutes . diluted in M-PER/protease inhibitor according to provided protocol Pipette 25µL of standards in technical duplicates into a 96-well clear assay plate Pipette 25µL of samples in technical duplicates into the assay plate. load an ice pack. Add 150µL of 1x SDS lysis buffer to each well. After allowing appropriate time for expression in transfected cells (usually 24 hours). 2.6 mL of 1x SDS lysis buffer. 4. 4. Boil all samples at 100˚C for 15 minutes 5. Cut two nitrocellulose membranes and incubate in Tris-Glycine buffer until ready to use. Cut edges of gel so that all lanes are included 3. and 20µL of protease inhibitor in a 15mL falcon tube 2. Load into apparatus. During procedures. one for αactin) 5. 4. 6. Whatman Gel Blot Paper. Make up MES buffer from 20x MES solution in milliQ H2O Load a 4-12% bis-tris gels into a vertical electrophoresis apparatus. 2. PVDF membrane. 5. Fill with MES buffer. Read plate at 260nm Calculate necessary volume for 1µg of total protein SDS-PAGE 1. Store at -80C. Load 5µL of each lysate solution into each well in collated duplicate (one for α-tau. Run gel at 200V for 35 minutes or until 10KDa marker is at the bottom of the gel Transfer 1. 6. 3. Make up albinum standards. 4. Whatman Gel Blot Paper. In the space provided. 2. In first lane. Prepare “sandwich” as follows: a. Combine 1. Gel. load 5µL of benchmark-prestained protein ladder.
10. 1:10. then 1x 7 minutes in TBS Block 45 minutes in 5% Milk – TBS/T with appropriate dilution of primary protein antibody (e. then 1x 7 minutes in TBS Block 45 minutes in 5% Milk – TBS/T with appropriate dilution of appropriate secondary antibody (e. 3. 10mL 1M Tris.g. 9. 1:8000 α-mouse) Wash 4x 7 minutes in TBS/T Place membrane between the two sheets of plastic of a clear page protector. . Make up ECL mix and pipette 1mL over each membrane. Expose for one minute and load into developer. 7. 4.TBS/T: 5% powdered milk in TBS/T Protocol: 1. 2. Rinse membrane TBS. load film into cassette. Block 45 minutes in 5% Milk – TBS/T with shaking Incubate 2x 7 minutes in TBS/T. Incubate 2x in TBS for 7 minutes with shaking. 5. Expose film to membrane longer as indicated by image on 1 minute exposure. In a darkroom.g.000 for mouse-α-tau and mouse-α-actin) Incubate 2x 7 minutes in TBS/T. 8. Tape page protector with membrane into a film cassette. 6.Blotting Buffers: TBS: Tris-buffered saline – 960mL milliQ H2O. 30mL 5M NaCl TBS/T: 999mL TBS + 1mL Tween-20 5% Milk .
4. 8. add 3mL of LB media and 1. In a falcon tube. Follow midiprep or maxiprep kit protocol to isolate plasmid from DH5α. 6. Bacterial Culture for Plasmid Preparation (midiprep. 5. maxiprep) 1. 2. 4.2µL of plasmid into 25µL of DH5α cells Incubate on ice for 20 minutes Incubate at 42C for 1 minute Incubate on ice for 2 minutes Add 200µL of SOC media. Move solidified LB-AMP agar plates to 32C incubator to warm before plating Add full volume of transformed bacteria to the solidified surface of an LB-AMP agar plate. Add 40µL (midiprep) or 140µL (maxiprep) of ampicillin. 2. Incubate at 32C with shaking for 45 minutes.Transformation of DH5α Cells 1. the Macherey-Nagel kits were used. Typically. Incubate at 32C for 12 hours. 3. Make up appropriate number of LB-AMP agar plates.5µL of ampicillin Touch a p10 micropipette tip to a single colony and drop into media Incubate tube at 32C with shaking for 8 hours Make up 80mL (midiprep) or 280mL (maxiprep) of LB media. 5. 3. Add 0. Spread evenly across plate with a sterile metal loop. . Incubate overnight at 32C with shaking 6. Transfer 3mL culture to 80 or 280mL LB-AMP solution. 7. Allow to cool.