Team:HKU-Hong Kong/Lab Protocol

1. Preparation of agarose gel
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.
2. Electrophoresis
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. Gel extraction
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.
2. DNA extraction (Adopt from Qiagen)
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, with the enzymes added at the last, into a tube

   The four binding reactions: Negative control, Positive control, Specific competitor and Non-specific competitor

2. All steps should be carried out on ice.
3. Mix well after addition of all the reagent.
4. Incubate the mixture at 37oC for several hours.

1. Centrifuge the sample at 8,000 rpm for 1 minute.
2. Discard the supernatant.
3. Add 250 ul P1 buffer to resuspend the pellet (tap to suspend the pellet completely).
4. Add 250 ul P2 buffer and mix gently by inverting the tube for several times.
5. Add 350 ul N3 buffer and mix thoroughly. The solution should now turn cloudy.
6. Centrifuge the solution at 13,000 rpm for 10 minutes.
7. Transfer the supernatant to a spin column with a collection tube inside.
8. Centrifuge at 12,500 rpm for 1 minute. Discard the flow through.
9. Add 750 ul PE buffer to the collection tube and centrifuge at 12,500 rpm for 1 minute.
10. Discard the flow through and centrifuge again to remove all remaining washing buffer.
11. Place the collection tube into a new eppendorf tube.
12. Add 50 ul elution buffer directly at the centre of the membrane of the collection tube.
13. Let it stand for approximately 3 minutes.
14. Centrifuge at 12,500 rpm for 1 minutes and collect the flow through (i.e. the product).

Colony PCR

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.

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. Add the following reagents, with the enzymes added at the last, into a tube.

   Composition for DNA ligation.

2. Incubate at 16oC overnight.

1. Send to BGI company for sequencing.

1. Pipette 3 mL of LB broth into a culture tube.
2. Add 3 ul of Ampicillin or 3 ul of Chloramphenicol.
3. Pick a single colony by a sterile pipette tip.
4. Place the culture tube in the rotary shaker and incubate at 37oC overnight.

1. Seed culture:
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.
2. Main culture:
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.
3. Aseptically transfer the cells to a sterilized, chilled 50-mL polypropylene tube and cool the cultures to 0oC by placing the tube on ice for 10 minutes.
4. Centrifuge at 4,000 rpm for 5 – 15 minutes at 4oC.
5. Decant the media from the cell pellets.
6. Resuspend the cell pellets in 20 mL of filtered, sterilized, chilled 0.1M calcium chloride (CaCl2).
7. Vortex gently to mix it and place the tube on ice for 15 to 30 minutes.
8. Centrifuge at 4,000 rpm for 5 minutes at 4oC.
9. Add 1 mL of chilled glycerol to each tube of culture.
10. Pipette up and down to mix it gently.
11. Add 100 ul culture in each eppendorf tube.
12. Store the culture at -80oC for approximately 1 hour.

1. Pipetting the liquid culture (about 200 ul) onto the surface of the LB agar plate.
2. Sterilize an L-shape glass rod.
3. Spread the cells evenly on the plate.
4. Incubate the plate at 37oC overnight with the bottom facing upward.

1. Sterilize the inoculating loop in flame.
2. Pick a portion of a single colony of the sample.
3. Make the first phase streak.
4. Flame sterilize the inoculation loop.
5. Cross the first phase of inoculum and make the second phase streak.
6. Repeat step d and e for making the third phase streak.
7. Flame sterilize the inoculating loop.
8. Incubate the plate at 37oC overnight with the bottom facing upward.

1. Mix 1 ul of DNA in 100 ul of competent cells.
2. Place the mixture on ice for 40 minutes.
3. Heat shock the cells in 42oC water bath for 90 – 100 sec.
4. Incubate on ice for 3 minutes.
5. Recovery:
1. Add 900 ul LB broth to the tube.
2. Incubate the mixture at 37oC for 1 hour in the rotary machine.
6. Spread 100 ul of each culture on a LB agar plate.
7. Incubate at 37oC overnight.

1. Add 1 g of ampicillin powder to 10 mL of ddH2O. Mix well.
2. Filter the solution using 0.22 um filter.
3. Store the filtrate at -20 oC.

1. Add 7 g of LB powder to 200 mL of ddH2O. Mix well.
2. Autoclave the solution.
3. Add 180 ul of ampicillin to 180 mL of molten agar, in a ratio of 1:1000 (if necessary).
4. Pour 10 mL agar solution per plate.
5. Let the agar solidify.

1. Add 4 g of LB powder to 200 mL of ddH2O. Mix well.
2. Autoclave the solution.
3. Add 200 ul of ampicillin to 200 mL of LB broth (1:1000).

1. Add 50 mL of 50X TAE buffer.
2. Add 2.5 L of ddH2O.

1. Gel Preparation
   1. Clean all the glassware by distilled water (ions-free).
   2. Prepare a non-denaturing 4% acrylamide gel according to the following formula:

      Formula for preparing acrylamide gel.

   3. Allow the gel to stand until the gel is completely polymerized.
2. DNA Binding Reactions
   1. Set up four binding reactions (if necessary) with the following composition.

      The four binding reactions: Negative control, Positive control, Specific competitor and Non-specific competitor

   2. Incubate the reactions at room temperature for 10 minutes.
   3. Add 1 ul of labeled DNA sample to each reaction.
   4. Incubate the reactions at room temperature for 20 minutes.
   5. Add 1 ul of 10X loading buffer for each reaction.
3. Electrophoresis
   1. Pre-run the gel in 0.5X TBE buffer for 10 minutes at 350V.
   2. Load the sample.
   3. Run the gel at 350V until the loading dye reached three fourth of the gel.
   4. Maintain the gel temperature under 30oC.