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Team:Bilkent UNAM Turkey/Notebook/Week7
From 2011.igem.org
Team:Bilkent UNAM Turkey/safety
From 2011.igem.org
10/08/2011
Today we start to making transformation of algae with glass beads method. Protocol is which we follow;
Grow cells in SGII+NH4NO3 until they reach a density of 1-2 x 10^6/ml. Spin down cells at moderate speed (5000 rpm for 5 min in a GSA type rotor). Resuspend in 1/100 volume SGII+KNO3 and allow to shake at room temperature for 2-4 hours. If desired, treat cells with gamete autolysin (prepared as described in The Chlamydomonas Sourcebook*) for approximately 45 minutes. I usually resuspend the cells in about 1/25 the original volume in autolysin. Spin and wash once with SGII+KNO3 before transformation. Optional: Test effectiveness of lysis by sensitivity to 0.05% NP-40. (Count duplicate samples +/- detergent in hemacytometer.) Note: Several recent experiments suggest that autolysin treatment increases the transformation rate of cw-15 mutants. Work in lots of a few tubes at a time. Add 300 microliters cells, 100 microliters 20% polyethylene glycol, 1-2 micrograms DNA (linearized DNA generally transforms somewhat more efficiently than supercoiled), and 300 mg sterile glass*** beads. Vortex for 15-30 seconds at top speed on a Fisher Vortex Genie 2 mixer; plate cells immediately on an SGII+KNO3 selective agar**. Depending on age and moisture of plates, parafilm immediately or after 24 hours; and put in the light. Colonies should be visible in about 6 days. Compounds of soluntions; 2000 mL SGII-NH4 medium: NaH2PO4: 7.34 g. K2HPO4: 2.3 g. Na acetate (NaC2H3O2 · 3H2O): 4 g. 20 mL of 5% Na-citrate. 20 mL of 0.1% FeCl3 (must be freshly made). 20 mL of 0.4% CaCl2 · 2H2O. 20 mL of 1.5% MgSO4. 20 mL of 3% NH4NO3. 20 mL of trace elements stock. Divide into appropriate quantities (see Plant Material) and cover with a cotton or foam plug and foil. Autoclave. Reserve 600-700 mL for making agar plates. Additional medium will be required if Part II (optional) is completed. 1000 mL Trace element stock. One solution containing: H3BO3: 100 mg. ZnSO4 · 7H2O: 100 mg. MnSO4 · 4H2O: 40 mg. CoCl2 · 6H2O: 20 mg. Na2MoO4 · 2H2O: 20 mg. CuSO4: 4 mg. 25 plates To 700 mL of the SGII-NH4 medium add 10.5 g of agar (to make 1.5% agar). Autoclave and allow the medium to cool somewhat before pouring. Pour into sterile petri dishes under sterile conditions; if possible let the dishes cool under sterile conditions before capping to prevent excessive condensation. 1000 mL 2X concentration of SGII-NO3 medium. This medium contains the same components as the recipe for SGII-NH4 medium but uses KNO3 instead of NH4NO3. NaH2PO4: 7.34 g. K2HPO4: 2.3 g. Na acetate (NaC2H3O2 · 3H2O): 4 g. 20 mL of 5% Na-citrate. 20 mL of 0.1% FeCl3 (must be freshly made). 20 mL of 0.4% CaCl2 · 2H2O. 20 mL of 1.5% MgSO4. 20 mL of 10% KNO3. 20 mL of trace elements stock. Autoclave. Dilute 250 mL with distilled water to make 500 mL of regular strength SGII-NO4 medium (400 mL for rinsing the agar plates and 100 mL for resuspending cells). Use the remaining 750 mL of 2X concentration medium to make agar plates. Additional medium will be required if Part II is done. 50 plates SGII-NO3 medium and 1.5% agar. Rinse the agar beforehand to remove ammonium by placing 7.5 g agar in each of three graduated bottles and bringing to 500 mL with distilled water. After the agar has settled, replace the water and shake; repeat once more, then bring to 250 mL with distilled water. Finally, add sufficient 2X SGII-NO3 medium to bring to 500 mL. Autoclave and let the medium cool before pouring, then pour into sterile petri dishes.Under sterile conditions, rinse the surface of the gelled agar with sterile SGII-NO3 medium. 100 mL 20% (wt/vol) polyethylene glycol (MW = 8000). Autoclave. 200 mL 70% ethyl alcohol. We are working on obtimization of this protocol. Funny part is when I look at algae under microscopy it is dancing and really amazing I will try to find a video for you. |
