Team:HKU-Hong Kong/Lab Protocol

1. Pour 100 mL of 1X TAE buffer into a conical flask.
2. Add the agarose powder to the buffer in the amount with respect to the concentration of the agarose solution (e.g. add 1 g for preparing 1% agarose gel solution).
3. Use a plastic wrap to cover the opening of the conical flask and microwave for approximately 2 minutes or until the agarose dissolves completely.
4. Pour the agarose solution into another conical flask which specifies for holding ethidium bromide (EB) – containing solution.
5. Add 1 – 2 ul of EB into the agarose solution and mix well.
6. Pour the solution into a gel tray with a comb. Remove any bubbles formed.
7. Allow the gel to solidify which takes approximately 30 minutes.
8. Discard all the wastes into the EB waste box.

1. Remove the comb and place the solidified gel into the electrophoresis tank.
2. Add TAE buffer to the tank when necessary.
3. Add 6X loading buffer to the DNA sample in the ratio of 1:6 and mix well.
4. Load the samples into the wells with care.
5. Load 2-3 ul of marker to a well for reference.
6. Run the electrophoresis at around 140V for about 30 minutes.
7. Take the gel photo in the UV-illuminating machine.

1. Wear UV protection glasses before the gel extraction.
2. Place the gel onto the transilluminator.
3. Turn on the transilluminator and quickly cut the desired gel band.
4. Place the cut band into an eppendorf tube for further processing.
5. Discard all the wastes into the EB waste box.

1. Weight the Eppendorf tube and determine the weight of the cut band.
2. Add 3 volumes of extraction buffer to 1 volume of the cut get.
3. Place the Eppendorf tube (with the cut gel and the extraction buffer) into 55oC water bath to dissolve all the agarose gel.
4. After dissolving, add the mixture to the spin column with a collection tube.
5. Centrifuge at 11,000 rpm for 1 minute.
6. Discard flow through.
7. Add 750 ul of washing buffer and centrifuge at 11,000 rpm for 1 minute.
8. Discard the flow through and centrifuge again at 11,000 rpm for 1 minute.
9. Place the collection tube to a new Eppendorf tube.
10. Add 20 mL elution buffer directly to the centre of the membrane of the collection tube.
11. Let it stand for approximately 3 minutes.
12. Centrifuge at 11,000 rpm for 1 minutes and collect the flow through (i.e. product).
13. Take a small portion of the DNA product for confirmation by gel electrophoresis.

1. Add the following reagents into a PCR tube (in order) and mix well.
2. Set the following PCR program.

Reverse PCR

1. Add the following reagents into a PCR tube (in order) and mix well.
2. Set the following PCR program.

Overlap PCR

1. First, two PCR reactions are set for amplifying the two genes, tetR and HNS, separately.
2. Add the following reagents into a PCR tube (in order) and mix well.
3. Set the following PCR program.
4. Set up another PCR reaction using a primer with a linker to link the two genes.
5. Add the following reagents into a PCR tube (in order) and mix well.
6. Set the following PCR program.
7. Set up another PCR reaction to further amplify the fused product.
8. Add the following reagents into a PCR tube (in order) and mix well.
9. Set the following PCR program.

1. Pick a single colony from a plate with fresh grown cells (for 16 – 20 hours at 37oC) and transfer it into 3 mL of LB broth in a sterilized 15-mL polypropylene tube.
2. Incubate the culture overnight at 37oC in a rotatory shaker to provide vigorous shaking.

1. Inoculate 1,000 ul of seed culture into 100 mL of LB broth in a sterile 250-mL flask.
2. Incubate the culture at 37oC with vigorous shaking (in a rotary shaker) for approximately 2 hours or until the OD600 value reaches 0.3 to 0.4.

1. Add 900 ul LB broth to the tube.
2. Incubate the mixture at 37oC for 1 hour in the rotary machine.