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Tina:LabNotes/2012-4-12
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*'''Preparation of Matrigel aliquots''' **Material: BD Matrigel hESC-qualified matrix (5 mL/vial) **catalog number: 354277 **Lot number: 17598 **Dilution factor: 290 uL (Mix 290 uL of Matrigel with 25 mL of DMEM/F-12 to coat twenty-four 35 mm dishes) **Everything contact Matrigel should be cooled. 1. Thaw Matrigel stock solution at 4°C, overnight. 2. Pre-chilled 1.5 mL eppendorf tubes, 15 mL conical tubes and an unopened box of 1000 uL tips at -20°C, overnight. 3. Put Matrigel and 1.5 mL tubes on ice. 4. Aliquot 56 uL of Matrigel stock solution to a total of fifty 1.5 mL eppendorf tubes as quick as possible. Switch tips every 5 tubes to ensure sterility and that the tips are chilled. 5. Aliquot 148 uL of Matrigel stock solution to a total of twelve 15 mL conical tubes. 6. Store aliquots at -70°C (for up to 6 months). *'''Preparation of Matrigel plates''' **Tissue culture-treated plates should be used. ** Matrigel plates are good for 1 week at 4°C, although fresh plates are preferable. Plates must be warmed to RT before use. 1. Pre-chilled sterile 1000 uL tips and 15 mL tubes at -20°C, overnight. 2. Put Matrigel aliquot, DMEM/F-12 and a 15 mL tube on ice. 3. Add 745 uL of ice-cold DMEM/F-12 to the Matrigel aliquot and then transfer to a 15 ml tube. 4. Add an additional 4 mL of ice-cold DMEM/F-12 to the 15 ml tube. Pipet gently to avoid bubbles formation. 5. Add 1 ml of the Matrigel working solution to four 35 mm culture plates and rock gently to distribute the solution evenly. 6. Note the date when Matrigel was plated on each plate lid. Seal the edges of the plates and put them at 4°C fridge.
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