Experiments

Molecular Biology Techniques

Gibson Assembly Protocol

1. Design fragments (G-Blocks) and order the necessary primers to open destination plasmid and prep plasmid for Gibson Assembly.
2. Digest with DPN1 to remove the backbone (1uL for overnight).
3. Perform Gibson Assembly Reaction.
   Note: All components should be kept on ice.

4. Incubate in a thermocycler at 50ºC for 1 hour.
   Note: Time of incubation will vary based on size and number of fragments.
5. Run on agarose gel to check size.
6. Transform into competent E.Coli cells.

Polymerase Chain Reaction (PCR) Protocol

Primer Reconstitution

1. Spin lyophilized primer vials at 6800G for 3 minutes.
2. Resuspend primers to a final concentration of 100nM.
3. Prepare 10 nM stock of both forward and reverse primers by diluting with DI water.

Prepare 50 µL PCR Reaction Mix

Follow the table for making the PCR reaction mix.

Amplification of Desired Segment using Thermocycler

Follow the table to amplify the DNA segment with a Thermocycler.

*Will vary depending on primer

Purification and Quantification

1. Purify using Monarch PCR Purification Kit.
2. Elute 30 µL into an eppendorf tube.
3. Nanodrop purified product by adding 1 µL of the sample.
4. Record concentration (ng/µL), A260/A280 score, and A260/A230 score.
   Note: Blank nanodrop first with the elution buffer to calibrate.

Colony Polymerase Chain Reaction (cPCR) Protocol

Primer Reconstitution

1. Spin lyophilized primer vials at 6800G for 3 minutes.
2. Resuspend primers to a final concentration of 100nM.
3. Prepare 10 nM stock of both forward and reverse primers by diluting with DI water.

Pick Colonies

1. Add 100 µL of nuclease free water to an eppendorf tube.
2. Pick large, well isolated single colonies and inoculate nuclease free water.

Prepare 50 µL cPCR Reaction Mix

Follow the table for making the cPCR reaction mix.

Amplification of Desired Segment using Thermocycler

Follow the table to amplify the DNA segment with a Thermocycler.

*Will vary by primer

Run Product on DNA Gel

1. To determine if appropriate DNA was inserted and amplified, run cPCR reaction on DNA Gel following DNA Gel Electrophoresis Protocol.

Cloning and Transformation

Making Overnight Cultures Protocol

Plate

1. Pick one colony from the chosen transformation plate using a 10 µL micropipette tip.
2. Place the micropipette tip into an eppendorf tube containing 100 µL of nuclease free water to resuspend the colony.
3. Add 5 mL of LB broth to a 15 mL Conical tube along with the appropriate antibiotics.
4. Take 50 µL of the nuclease-free water and colony mixture and add it into the 15 mL conical tube.
5. Take the other 50 µL of the water and colony mixture and plate it on the appropriate antibiotic plate.
6. Store the overnights and plates on the 37ºC.
7. Place all leftover water and colony mixture into the fridge. Labeled with the appropriate colony designation and the date.

Glycerol Stock

1. Add 5 mL of LB Broth plus antibiotics to a 15 mL conical tube.
2. Using a 5 mL glass pipet, stab the glycerol stock and transfer a shard of the stock to the LB.
3. Incubate overnight at 37ºC.

Plasmid Preparation Protocol

Following Monarch Spin Plasmid Miniprep Kit from New England BioLabs

1. Pellet 1-5 mL (not to exceed 15 OD units) bacterial culture by centrifuging at 16000G for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μL of B1 Resuspension Buffer. Vortex to ensure suspension.
3. Add 200 μL of B2 Plasmid Lysis Buffer and invert. Let sit at room temperature for one minute.
4. Add 400 μL of B3 Neutralization Buffer, invert a few times, and let sit at room temperature.
5. Centrifuge at 16000G for 3 minutes.
6. Transfer supernatant to labeled spin column and centrifuge again at 16000G for 1 minute. Discard flow-through.
7. Re-insert the column into the collection tube and add 200 μL of Plasmid Wash Buffer 1. Centrifuge at 16000G for one minute and discard flow-through.
8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for one minute.
9. Transfer column to a clean 1.5 mL micro-centrifuge tube.
10. Add 30 μL of DNA Elution Buffer to the center of the matrix, and then wait one minute before centrifuging at 16000G for 1 minute.
11. Use the NanoDrop machine to check the concentration of DNA in the final eluted DNA.

Transformation Protocol

1. Thaw 50 µL of competent BL21DE3 cells and add 2 µL of DNA into competent cells. Incubate on ice for 30 minutes.
2. Heat shock mixture in heat-block at 42ºC for 30 seconds and put back on ice for 2 minutes.
3. Preheat Super Optimal Media with Catabolite Repression (SOC) media to 37ºC. Add 950 µL of SOC to a final volume of ~1000 µL.
   Note: This step should be done in a sterile Biosafety Cabinet (BSC)
4. Incubate at 37ºC for 60 minutes. During this incubation period there needs to be aeration of some kind. For this, cells were put on a shake plate at 300 RPM.
5. Dry three LB plates with appropriate antibiotics in an incubator set to 37ºC. Plate 100 µL onto one plate and then plate 200 µL onto each of the remaining plates. Spread evenly around plate using a Colony Spreader.
6. Incubate at 37ºC overnight.

Gel Based Techniques

DNA Gel Electrophoresis Protocol

Make Gel

1. To make a gel add 1G of agarose to 100 mLs of 1X TAE in a glass beaker.
2. Microwave on high until dissolved. The solution should appear clear, if there is any streakiness or cloudiness keep microwaving.
3. For 100 mLs usually takes about 3 minutes.
4. Remove from the microwave with thick orange gloves and set to cool until you can comfortably touch with a bare hand.
5. Once you can comfortably touch the beaker with your bare hands, add in 1 μL of SYBR Safe stain for every 10 mLs of solution you have.
6. A 50 mL gel would get 5 μL of SYBR Safe.
7. Add a comb to a gel box and pour the gel into the gel box.
8. Make sure the gel box is assembled in such a way that the gel will not run out if you pour it in. The gel apparatus consists of a big open box with two electrodes and a smaller box with rubber seals and open on both ends. Make sure that the smaller box is put in on the elevated platform of the bigger box and with the open ends facing the walls of the bigger box. You should have to push hard as the rubber seals will drag.
9. When pouring the gel make sure that the gel covers the entire bottom of the box, if there are any open spaces, you have to add more.
10. Finally, when pouring the gel try to limit the number of bubbles in the path of the DNA.
11. If there are bubbles use a pipette tip to move them to the side.
12. Once gel has solidified (should be firm to touch), remove gel from orientation and rotate it 90 degrees. Remove the comb and pour in 1X TAE.
13. Make sure the gel is oriented correctly (as in the long part of the gel is facing towards the positive electrode).
14. Cover with enough 1X TAE so that the gel is covered, ensuring that the buffer goes into the wells.

Prep Samples

1. Portion out sample into a separate PCR tube.
2. Usually 5 uL of sample.
3. Add loading dye and mix thoroughly by pipetting up and down.
4. Loading dye will typically be at 6X concentration, so add 1 uL of dye to 5 uL of sample.
5. Create gel legend to keep track of the locations of the ladder and samples.
6. Make descriptive names so someone coming after you can understand which lane each sample corresponds to.

Load Gel

1. Load ladder and samples from left to right to ensure that you don't miss a sample or get confused.
2. Load 3 uL of ladder.
3. Load 6 uL of the samples.
4. Go in with the pipette very slowly, careful not to puncture the bottom of the gel.
5. Do not go past the second stop, this will expel air into the well and ruin your samples.
6. It can be helpful to use the other hand to steady the hand with the pipette
7. If your samples float away then you either need to add more loading dye or did not mix it sufficiently
8. To confirm ladder and samples were loaded properly, there should be a semi solid line of color at the bottom of the well.

Run Gel

1. Cover tank with gel cap.
2. There will be two wires coming out of it.
3. Make sure that the negative electrode is closest to the lanes.
4. Plug the tank into the power bank and run at 150 V for 30 minutes.
5. You can check that everything is working by seeing if bubbles appear in the 1X TAE.

Visualize Gel

1. Remove gel from box and place gel on gel imager to image.
2. Make sure that the gel imager is using the proper stain.
3. Export image and save.

Clean Up

1. The 1X TAE can be reused, so pour it into a 1 L bottle and label with the number of times it has been used.
2. Throw the gel away into biohazard waste.
3. If you accidentally made too many gels:
   Place the extra gels into a plastic bag and label with name, date, and gel stain + concentration (SYBR Safe, 1% Agarose).
   Add a little bit of 1X TAE into the bag.
   Store at 4°C.

Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) Gel Protocol

Sample Preparation

1. Combine the samples with 50 µL 10% PBS and 50 µL Laemmli.
2. Make Laemmli + PBS + B-mercapnoethanol mix by:
   50/50 of 10% PBS and 2x Laemmli + 5% B-mercapnoethanol
3. Resuspend samples in 100 uL of Laemmli mixture.
4. Place samples on a heat block for 10 minutes at 95°C.
5. Vortex all samples for 5 minutes.

Gel Set Up

1. Obtain Invitrogen Gel:
2. Remove packaging of gel.
3. Remove tape from the bottom of the gel.
4. Remove the comb from the top of the gel.
5. Rinse gel thoroughly with DI water.
6. Carefully insert gel into the tank.
7. Fill up the tank with 1x SDS buffer until the fill line.
8. Load ladder and samples carefully.
9. Run gel for 1 hour at 150V.

Staining

1. Remove gel from the tank carefully and remove from packaging
2. rinse several times in DI water.
3. Add 50 mL of Coomassie stain to the gel and cover with tin foil and leave it to stain overnight while rocking.

Making Agar Plates Protocol

1. Prepare LB Agar media. Mix LB Agar (Miller) power with DI water. Sigma-Aldrich LB Broth with Agar powder was used for plates.
   Note: 40g of LB agar powder per 1 liter of DI water.
2. Sterilize and dissolve LB-Agar mixture by microwaving in 30 second increments until powder is completely dissolved, swirling container between each increment until clear.
   Note: Autoclaving can also be used for this step: 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

The following steps should be done in a sterile Biosafety Cabinet (BSC):

3. Allow mixture to cool for 5 minutes and add sterile antibiotic, swirl container to mix:

5. Before LB Agar mixture is completely cool, pipette 12 mL of mixture into each plate. Allow plates to set without lids on to avoid condensation.
6. Plates can be stored at 4ºC until use.

Analytical Techniques

Protein Purification Protocol

Prep the Cellulose

1. Add Microcrystalline Cellulose (0.2g), along with distilled water (0.6mL). Mix lightly in beaker in order to create a slurry.
   Note: The cellulose will not dissolve, just create a slurry.
2. Slowly add 86% Sulfuric Acid (10 mL) with stirring. Let the solution sit on ice for 1 hour with occasional stirring.
3. Add 40 mL of ice-cold water gradually with stirring. This should result in a whitish, cloudy precipitate.
4. Using a swing bucket rotor, centrifuge the cellulose at 3,000G for 10 min at 4°C.
5. Decant the supernatant.
6. Resuspend in cold water and then centrifuge again at 3,000G for 10 min at 4°C.
7. Repeat the previous step four times.
8. If needed you can resuspend in 45 mLs of DI water and store at 4°C.
9. After the final wash measure the pH of the solution and if it is under 5 resuspend in 0.5 mLs of Na2CO3 (2M), along with 45 mLs of water.
10. Centrifuge at 3,000G for 10 minutes at 4°C and resuspend in DI water.
11. Remeasure the PH of the solution and repeat 9 and 10 until the solution reaches a pH of 5-7.
12. Resuspend the final solution in 40 mLs of DI water and store at 4°C.

Protein Purification

1. Transfer cultures into 50 mL conical tubes.
2. Harvest culture by spinning at 6800G for 5 minutes.
3. Decant supernatant.
4. Resuspend each culture in 15 mLs of 0.5% Tween20.
5. Place the conical tube in an incubator to shake for 10 minutes.
6. Combine 10 mLs of the NaOH + cellulose solution with 10 mLs of the lysate solution in a beaker with a stir bar.
7. Place the beaker on a stir plate for 10 minutes.
8. Transfer to another 50 mL conical tube and spin down at 14000G or max speed for 10 minutes.
9. Add 10 mLs of ethylene glycol and shake in hand for 2 minutes.
10. Spin down again at 14000G for 10 minutes.
11. Transfer supernatant into fresh 50 mL flasks and store at -20°C with the correct label.

Protein Growth Curve Protocol

1. Take cultures out of the incubator at 37°C.
2. Make flasks with 47.5 mLs of LB and 50 µL of Kan50.
3. Add 2.5 mLs of cultures to each of the flasks.
4. Put all the flasks in the 37°C incubator.
5. Grow until an Optical Density at 600 nanometers (OD600) of 0.6.
6. Induce half of the flasks with 1 mM IPTG and the other half with 250 µM IPTG.
7. Place half of the flasks in at 32°C.
8. Grow for three hours and record OD600.
9. Take 1 mL samples every hour to make protein growth curves.
   • X-axis: Time (Minutes)
   • Y-axis: Optical Density at 600 nanometers (OD600)

Burning Assay Protocol

Prep the DNA

1. Plasmid prep the DNA and elute in water.
2. Nanodrop the DNA.
   • Record concentration (ng/µL), A260/A280 score, and A260/A230 score.
3. If multiple preps were completed, combine all into one vial and calculate the new concentration and the total amount of DNA,
4. In fresh eppendorf tubes, create four different conditions:
   • Water
   • 0.625% by weight DNA
   • 1.25% by weight DNA
   • 2.5% by weight DNA
5. For example; if the DNA solution was 92.4 ng/μL
   • Condition 1: 560 μL water
   • Condition 2: 26 μL DNA solution -> 534 μL of Nuclease free water
   • Condition 3: 53 μL DNA solution -> 507 μL of Nuclease free water
   • Condition 4: 107.8 μL DNA solution -> 452.2 μL of Nuclease free water

Apply to Cotton

1. Take cotton and cut into strips ⅝ inch wide.
2. Weigh the cotton before adding.
3. Holding cotton with a pair of plies with one hand use a pipette to dot the DNA solution onto the cotton swab, adding just a thin layer. Cover both sides with the solution.
4. Place in a petri dish and label and leave on the bench overnight to dry.