Difference between revisions of "Team:FAFU-CHINA/Protocol"

Protocol of Polymerase Chain Reaction

Protocol of C. reinhardtii Transform

1.Measure the optical density of the C. reinhardtii cultures at 750nm (OD₇₅₀)

Note: For best performance, the OD750 of cultures should be between 0.3–0.5. If the OD750 does not reach 0.3 within 24 hours of incubation after dilution, incubate the cells for an extra 3–5 hours to allow for an additional cell division.

2.Harvest 15mL of the cell (per transformation) by centrifugation at 2,500 rpm for 10 minutes at room temperature. Centrifuge the cells in 15-Ml conical tubes to obtain tight pellets.

3.Discard the supernatant by decanting. Remove the remaining supernatant using a pipette.

4.Resuspend the cells in 250 μL of TAP-40 mM sucrose solution at room temperature by gently pipetting up and down.

5.Add 2 μg linearized plasmid DNA (i.e., pChlamy_3 construct containing your gene of interest) into the resuspended cells. Mix the DNA-cell suspension gently by flicking the tube. In a separate tube, prepare a control transformation with the pChlamy_2/Control Vector, linearized using PvfI restriction enzyme.

6.Transfer 250 μL of the transformation mixture into an electroporation cuvette and incubate at room temperature for 5 minutes.

7.While the transformation mixtures are incubating, add 5 mL of TAP-40 mM sucrose solution at room temperature into each well of a 6-well plate.

Note: You will divide each transformation mixture between two wells of a 6-well plate after electroporation, so that the cells in each transformation mixture will recover in 10 mL of TAP-40 mM sucrose solution total.

8.Set the electroporation parameters as follows:

9.Gently tap the electroporation cuvette to mix the contents and resuspend the settled cells, and place the cuvette in the cuvette chamber.

10.Electroporate the cells with the above parameters.

11.Split the transformation mixture into two aliquots of 125 μL each and transfer each aliquot into one well of the 6-well plate containing 5 mL/well of TAP-40 mM sucrose solution at room temperature. Wash the cuvette with 1 mL of TAP-40 mM sucrose solution to get most cells out of the cuvette and split and add the wash into the same two wells.

12.Place the 6-well plate in the algal growth chamber set to 26°C and 50 μE m–2 s–1.

13.Incubate the cells for 24 hours with gentle agitation (100–150 rpm) to let them recover.

14.Centrifuge the cells at 2,500 rpm for 10 minutes at room temperature.

15.Discard the supernatant by decanting. Remove the remaining supernatant with a pipette.

16.Resuspend the cells with gentle pipetting in 150 μL of TAP-40 mM sucrose solution at room temperature.

17.Plate the entire cell solution from each transformation on one TAP-agar-Hygromycin plate using disposable cell spreaders or glass plating beads to spread the cells evenly. Make sure the plates do not have condensation on them.

18.Place the plates agar side at the bottom in the algal growth chamber set to 26°C and 50 μE m–2 s–1. Do not stack the plates to ensure continuous and even illumination.

19.Incubate the plates for 5 days or until C. reinhardtii colonies are clearly visible. Control vector should produce a minimum of 30 transformants per electroporation reaction. The transformation efficiency with the pChlamy_3 construct will depend on the nature, size, and codon content of the gene of interest, and the physiological state of the cells.

20.Proceed to determination of integration by colony PCR (see page 16) before selecting clones for further scale-up. About 20% of the colonies should be positive for the gene of interest. Due to random integration and silencing events in C. reinhardtii, we recommend picking at least 10 positive colonies and testing them for the expression level of the gene of interest by RT-PCR (or Western blotting, if you have the antibody to detect it).