This procedure is used to engineer the ADP1 chromosome using exogenous DNA.

1. Grow an overnight culture of the ADP1 strain you are manipulating.
2. Prepare two culture tubes with 500 μL LB media + 35 μL of overnight culture. Label one tube “- DNA” and the other “+ DNA”.
3. Add 50-100 ng of the transformant DNA to the tube labeled “+ DNA.”
4. Incubate the tubes at 30°C and shake at 200 rpm overnight.
5. Dilute the “+ DNA” mixture 100-fold in sterile saline and plate 50 μL onto selective media. Repeat for the "- DNA" mixture.
6. Incubate plates overnight at 30°C.
7. Select 2-3 colonies from the “+ DNA” plate, and inoculate an overnight culture. Incubate at 30°C and 200 rpm.
8. Using the overnight culture, make glycerol stocks of each biological replicate.

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1. Obtain a standard culture tube for each strain to be inoculated, and an additional tube for the negative control.
2. Label one tube “- Control” and the other tubes according to the strain that they will contain<./li>
3. Add 5 mL of LB to each of the tubes.
4. If the growth medium requires an antibiotic, add 5 μL, (1000x of the stock concentration), of desired antibiotic to the culture tube.
5. Pipette 2 μL of a glycerol stock into each tube except for the - Control tube. Vortex briefly. If inoculating from a plate, select a single colony using a sterile loop and swirl the loop in the media.
6. Place the tubes in either a 30°C (ADP1) or 37°C (E. coli ) incubators at 250 rpm and shake overnight.

1. Grow an overnight culture of the strain you wish to freeze.
2. Obtain and label cryovials with the name of the strain that will be frozen.
3. Working around a lit Bunsen burner, add 200 μL of 80% Glycerol to the cryovial. Glycerol is thick, so use a P1000 pipette. Pull and push the pipette trigger slowly when extracting and dispensing the liquid.
4. Using an P1000 pipette, add 1 mL of overnight culture to the labeled cryovial.
5. Close the cryovial and vortex vigorously for ~10 seconds.
6. Place the cryovials in a -80°C freezer.

1. For a 50 μL reaction, pipette the following reagents into a 1.7 mL microcentrifuge tube to create a master mix:
• 2.5 μL of 10 μM upstream forward primer
• 2.5 μL of 10 μM downstream reverse primer
• 10 μL of 5x Phusion buffer
• 1 μL of 10 mM dNTPs
• 1.5 μL of Phusion Buffer
• 0.5 μL of Phusion Polymerase
• 32 μL of ddH₂O
• NOTE: The above quantities can be multiplied if more than one PCR needs to be performed. For example, a master mix for 3 PCRs would include 3 times the amount of each reagent.
2. Pipette 49 μL of master mix into a PCR tube.
3. Pipette 1 μL of template DNA into the PCR tube. For control tubes, pipette 1 μL of ddH₂O instead.
4. Place the PCR tube in the thermocycler and run the following conditions
1. Denaturation: 98°C, 0:10
2. Annealing: Tm (melting temperature) of Primers, 0:30
3. Extension: 72˚C, 0:30
4. Goto 1, 34x
5. 72˚C, 5:00
6. 12˚C, ∞

Making the Gel

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1. Add 50 mL of TAE buffer to a 250 mL Erlenmeyer flask.
2. Weigh out .5 grams of Agarose (1% Agarose Gel) and pour it into the Erlenmeyer flask.
3. Microwave the mixture for 1:30.
4. Add 2.5 μL (0.005%) of Invitrogen’s SYBR Safe DNA Gel Stain to the heated mixture.
5. Swirl the mixture to disperse the dye.
6. Pour the mixture into an agarose gel mold containing the desired number of wells.
7. Wait ~20 minutes for the mixture to solidify.
8. Once the gel has solidified, place the gel into the gel rig and pour TAE buffer until the gel is submerged.
9. Proceed to “Loading the gel.”

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Loading the Gel

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1. Add 6 μl of 1 kb+ ladder to the leftmost well.
2. Add 1 μl of NEB purple loading dye to 5 μl of PCR product and pipette up and down to homogenize the mixture.
3. Repeat step 2 for all samples.
4. Once all samples have been properly prepared, load each sample into the gel starting from left to right, noting which sample goes into each lane.
5. After each sample has been loaded, proceed to “Running the Gel.”

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Running the Gel

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1. Once all samples have been loaded, attach the lid to the gel rig and connect it to a power supply.
2. Set the power to 120 V and let the gel run for 20-30 minutes. (Note: 25-30 minutes is ideal for products longer than 2 kb because it allows for greater separation of the ladder)
3. After 20-30 minutes, pause or turn off the power supply, unload the gel tray, and place the gel into a transilluminator.
4. Proceed to "Analyzing the Gel."

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Analyzing the Gel

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1. Place the tray in a transilluminator and run a protocol that is compatible with Invitrogen's SYBR Safe dye.
2. Save the image of the gel.

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1. Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls.
2. Thaw tubes on ice.
3. Add transforming DNA (up to 25 ng, or 0.5-2 μL of a miniprepped plasmid) and incubate on ice for 30 minutes.
• For transforming cloning reactions (e.g. from Gibson Assembly), it is possible to add up to 5 μL of reaction directly to the cells without any purification steps.
4. Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds.
• If using a heat block, make sure to fill the wells with diH2O to ensure efficient heat transfer.
5. Immediately transfer tubes to ice, cool 1-2 minutes.
6. Add 450 μL SOC medium to each tube, and shake at 200-250 RPM for 1 hour.
• For transformations in Eppendorf tubes, place the tubes inside an empty 250 mL flask, and incubate in the large shaking incubators.
7. Plate at least two different volumes (10 μL and 50 μL) of each reaction on appropriate antibiotic plates.
• If your efficiencies tend to be on the low side, pellet your cells by centrifuging for 5 minutes at 3000 rpm. Resuspend in 100 μL of saline, and plate the entire mixture.

This procedure is used to determine the transformation efficiency and detction threshold of ADP1 .

1. Fill each of the wells of the 96-well plate that you will be using with 180 µL Saline/PBS (see image on right).
2. Fill the top row of your plate with 200 µL sample.
3. Using the P200 pipet, perform serial dilutions of 20 µL into each well (20 in 200 = 1:10 dilutions) - Change tips between each transfer!
4. Using your P20 pipette - pipette up to 5 µL of sample, line it up with the grid on your plate, touch the pipette tips to the plate, and slowly depress the plunger to deposit a row of droplets onto the agar.
• If your sample spreads into another sample, your plate is not dry enough! Stop what you are doing and let the plates dry.
5. Plate all your samples in triplicate from the same set of dilutions.
6. Allow your spots to dry, and transfer them to the appropriate incubator to grow.