Experiment Protocols

Transformation

1. Prepare 100 μL 1×KCM by adding 80 μL double distilled H2O and 20 μL 5×KCM into an EP tube and store it in an EP tube.
2. Add 10 μL of the prepared plasmid solution into the EP tube and place it on ice.
3. Take competent cells from -80 degrees storage and thaw in ice for 2-3 minutes.
4. Put 100 μL of competent cell into the EP tube, place on ice for 30 minutes.
5. Take out the EP tube and put it into a 42 degrees water bath for 90 seconds.
6. Take out the EP tube from the water bath and immediately place it on ice and wait for 3 minutes.
7. Add 800 μL of liquid LB (without the desired antibiotic) into the EP tube.
8. Put the EP tube into incubator shaker of 37 degrees for 40 minutes with a speed of 220-240 rpm.
9. Take out the EP tube and put it into a centrifuge with a speed of 12000g for 30 seconds.
10. Take the EP tube from the centrifuge and proceed the following steps in a laminar flow clean bench.
11. Extract and dispense 800 μL of supernatant (when inserting the pipette into the EP tube, do not touch the condensed solid at the bottom).
12. Pipette up and down (with a smaller pipette tip) to mix the remainder of the solution inside the EP tube. Avoid creating bubbles, so mix gently, but thoroughly until nothing remains at the bottom.
13. Using a pipette, deliver 200 μL of the solution onto the LB petri dish (containing the desired antibiotic, which can be added in a 1:1000 ratio with the LB media prior to its solidification on the petri dish).
14. Slightly burn another pipette tip on fire for 2 seconds, then bend it.
15. With the bent pipette tip, spread the solution on the petri dish.
16. Close the petri dish lid and write down the sample name and the date this was performed.
17. Flip the petri dish upside down and put into a 37 degrees constant temperature and humidity incubator. Wait for 16-18 hours.

Plasmid Miniprep (Spin) (as provided by Axygen and modified)

1. Collect 1-4 ml of overnight LB culture (depending on the amount of bacteria grown). Centrifuge at 12,000×g for 1 minute to pellet the bacteria. Decant or pipette off as much of the supernatant as practical.
2. Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing.
3. Add 250 μl of Buffer S2, and mix by gently inverting the tube for 4-6 times.
4. Add 350 µl of Buffer S3, and mix by gently inverting 6-8 times. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
5. Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000×g for 1 minute.
6. Pipette 500 µl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
7. Pipette 700 µl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
8. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column and centrifuge at 12,000×g for 1 minute.
9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. To elute the purified plasmid DNA, add 50 µl of deionized water to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.

Transformation into Agrobacterium

1. Competent cells rest (from -80 °C) in ice for 2-3 minutes to melt.
2. Add plasmid (5-10 mL, depending on concentration) into competent cells.
3. Pipette up and down to mix properly.
4. Rest on ice for 30 minutes.
5. Put into liquid nitrogen for 5 minutes.
6. Put into water bath of 37 °C for 5 minutes.
7. Add 700-800 mL of YEB.
8. Put into incubator shaker of 28 °C for 8 hours.
9. Extract 200-250 mL to spread on petri dish (YEB + Agar).
10. Put the rest in test tube with 5 mL YEB.

Agrobacterium-mediated Transformation

1. Calibrate and dilute the agrobacteria solutions to an OD600 of 1.0.
2. Centrifuge the agrobacteria solutions at 12000 g for 1 min, and remove the supernatant.
3. Resuspend the agrobacteria in a 0.01 mol MgCl2 solution.
4. Add an acetosyringone solution, making a final solution concentration of 0.2 M.
5. Leave aside for 3 hours at 25-28 °C in order to fully activate the agrobacteria cells.
6. Use a disposable syringe and inject the solution into the plant’s leaves. [Note: this can be done by placing a finger under the leaf at the position of the syringe, and gently pressing against the syringe tip while injecting the solution.]

Gel Extraction (as provided by Axygen and modified)

1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. In this application, the weight of gel is regarded as equivalent to the volume. Transfer the gel slice into a 1.5 ml microfuge tube.
2. Add a 3x sample volume of Buffer DE-A into the microfuge tube.
3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved. Intermittent vortexing (every 2-3 minutes) will accelerate gel solubilization.
4. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix.
5. Place a Miniprep column into a 2 ml microfuge tube. Transfer the solubilized agarose from Step 4 into the column. Centrifuge at 12,000×g for 1 minute.
6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μL of Buffer W1. Centrifuge at 12,000×g for 30 seconds.
7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μL of Buffer W2. Centrifuge at 12,000×g for 30 seconds.
8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μL aliquot of Buffer W2 and centrifuge at 12,000×g for 1 minute. (Note: Two washes with Buffer W2 are used to ensure the complete removal of salt, eliminating potential problems in subsequent enzymatic reactions, such as ligation and sequencing reaction.)
9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000×g for 1 minute. 10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube. To elute the DNA, add 30-50 μL of deionized water to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.

Gradient test

1. Culture E. coli with desired plasmid (e.g. pSB1C3-Lux) in an Erlenmeyer flask with liquid LB overnight (around 16 hours).
2. Extract 5 mL of the solution into test tubes, and store the test tubes at 4 degrees Celsius.
3. At every desired time period (e.g. every 1 hour), take out a tube.
4. Inject 500 μL of L-Arabinose (with certain concentrations) into the tube.
5. Place the tube into a 30 degrees Celsius shaker.
6. Repeat steps 3 to 5 for every time period that is to be measured.
7. Take out all the tubes from the shaker.
8. Extract 100 μL from each tube into separate 96-well plate holes. Multiple repeats are recommended for each tube.
9. Place the 96-well plate into a microplate reader to read the absorbance and fluorescence.

PCR (50 μL)

1. Prepare all reaction components on ice.
2. Add 5 μL dNTPs, 5 μL buffer, 3 μL MgSO_4, 1.5 μL forward and reverse primer, 1 μL template DNA, 1 μL enzyme (depending on the DNA needed).
3. Add dd H2O so that there is in total 50 μL of solution.
4. Put in PCR machine.
5. For PCR of enzyme, set machine at 1kb/min.

PCR Machine Setting

1. Set machine at 1kb/min
2. Step 1: 98 °C for 10 minutes
3. Step 2: 35 cycles, 98 °C for 30 seconds, (TM-5)°C for 30 seconds, 72°C for x (1kb/min) minutes
4. Step 3: 72 °C for 7 minutes
5. Step 4: 16 °C for 10 minutes
6. ***For KOD enzyme, use 68 °C instead of 72 °C

Enzyme Digestion (50 μL)

1. 5 μL 10x buffer
2. 2 μg DNA
3. 1 μL enzyme (thermofisher) each
4. dd H2O up to 50 μL

Ligation (20 μL)

1. 1-5 μL plasmid and fragment (depending on concentration)
2. 1 μL T4 DNA ligase
3. 2 μL DNA ligase buffer
4. dd H2O up to20 μL
5. Incubate at 16 °C overnight

LB Preparation (1L)

1. Weigh 5g Yeast Extract, 10g NaCl, 10g Tryptone and add to beaker.
2. Add 16g Agar Powder for solid medium.
3. Add less than 1L of H2O into the beaker.
4. Add a stirring bar to autoclave until all is mixed.
5. Until compounds fully dissolve, fill up water to 1L.
6. Sterilize before use (under a condition of 121°C, 20 mins).

YEB Preparation (1L)

1. Weigh 1g Yeast Extract, 5g Tryptone, 5g Sucrose, 0.5g MgSO4 and add to beaker.
2. Add 16g Agar Powder for solid medium.
3. Weigh 5g Beef Extract (tare spoon and weigh with spoon).
4. Add less than 1L of H2O into the beaker.
5. Add a stirring bar to autoclave until all is mixed.
6. Beef Extract (on spoon) needs to be held during the stirring.
7. Until compounds fully dissolve, fill up water to 1L.
8. Sterilize before use (under a condition of 121°C, 20 mins).

Arabinose Preparation (1M, 50mL)

1. Weigh 3.00g Arabinose and add to a 50mL Corning tube.
2. Amount of Arabinose depends on molarity desired.
3. Add less than 50mL H2O into the tube.
4. Mix thoroughly.
5. Might require the help of a microwave if higher molarity is desired.
6. When Arabinose fully dissolves, fill up water to 50mL.
7. Store in 4°C fridge, and filter with a disposable syringe before use

Gel Electrophoresis

1. Add 100mL 1×TAE to an Erlenmeyer flask.
2. Weigh 1g Agarose and add to the flask.
3. Microwave 1 min for Agarose to fully dissolve in the 1×TAE solution.
4. Add 10μL Ethidium Bromide into flask (use disposable gloves).
5. Swirl the flask until it is mixed thoroughly with the TAE and Agarose.
6. Pour the mixture into the gel tray and insert a suitable comb (variable in size and number of wells).
7. Wait around 15 minutes until gel solidifies.
8. Add loading buffer to each DNA sample.
9. Make sure enough 1×TAE is in the gel tank.
10. Place the gel with the wells closer to the negative electrode. (Note: DNA will run towards the positive electrode)
11. Add the DNA sample carefully to the wells.
12. Add 10μL DNA marker to a well (preferably on the outermost well).
13. Run the gel electrophoresis (120V for 40 mins or 100V for 60 mins).