Team:Gaston Day School/Experiment

Experiments and Protocol


For MCF tubes:

  1. P: Plasmid Prep
  2. D: Digest
  3. T: Transformation
  4. L: Ligation


  1. x = the number of transformations you are doing
  2. Place the SOC medium (from the fridge) in the incubator
  3. Also place X plates in the incubator (With the appropriate antibiotic resistance)
  4. Set the heating block to 42°C
  5. Make an ice-water bath
  6. Obtain a float and X premade MCF tubes with 100µL competent cells from -80°C freezer
  7. Add 2µL of the DNA to competent cells
  8. Place it in the ice-water bath for 2 mins
  9. Place tubes in the heating block for 30 seconds
  10. Another 2 mins on ice
  11. Add 1 ml of warm SOC medium
  12. Place each tube in the incubator (37℃) for 1 hour
  13. Centrifuge to pellet bacteria, then pour off most of the supernatant
  14. Resuspend, then plate the cultures on the appropriate antibiotic

Pick Colonies

  1. Obtain a metal loop, sterile LB tubes, plates from either the fridge or incubator
  2. Label the tubes with corresponding plates
  3. Spot the colonies (Choose more than one if available)
  4. Flame sterilize the metal loop and use it to gently obtain the colonies
  5. Swirl around in the LB solution to make sure the colony detaches from the loop
  6. Put the tubes into the shaking incubator to grow overnight

Plasmid Prep Procedure

  1. TENS procedure (make fresh each time)-To 4.5 ml TE solution add 250 ul 10% SDS and 250 ul 2M NaOH
  2. Transfer 1.5ml bacterial culture to a labelled MCF tube
  3. Centrifuge for 30 seconds to pellet bacteria
  4. Pour off most of the growth medium. Leave approximately 100ul in the tube
  5. Resuspend the pellet by shaking, vortexing, or tapping the tube vigorously. Make sure the bacteria are completely resuspended and no clumps remain before continuing.
  6. Add 100ul TENS to the tube
  7. Mix well by inversion for 2 minutes. Do not shake the tube. The solution will get viscous
  8. Add 150ul sodium acetate and mix well
  9. Centrifuge for 3 minutes
  10. Transfer the supernatant (liquid) to a clean, labeled MCF tube
  11. Add 1mL 95% Ethanol to the tube and mix well by inversion. You may see DNA as faint white strands in the liquid. The tube should be nearly full of liquid. Sharpie brand pens have an ink that is soluble in Ethanol. Make sure your labels do not get removed accidentally.
  12. Centrifuge 5 minutes to pellet the DNA. Place the tubes in the centrifuge with the hinges out to make finding the pellet easier
  13. Pour off the 95% Ethanol
  14. Add 500μL 70% ethanol to and mix by inversion to wash the pellet
  15. Centrifuge again for 5 minutes
  16. Pour off the 70% ethanol
  17. Place the tubes upside-down and allow them to dry completely
  18. Resuspend the pellet in 20-30μL distilled, sterilized water
  19. Use 5 uL of the DNA in a restriction digest or 1 uL for PCR


  1. Obtain the plasmid prep tubes, new MCF tubes, enzymes in the freezer
  2. For each new tube, prepare the master mix(MM):
    • Distilled Waterr 12µL
    • CutSmart Buffer 2µL
    • Upstream Enzyme 0.5µL
    • Downstream Enzyme 0.5µL
  3. Each tube needs 15µL of the MM, in order to get enough MM into each tube. If you have x digest, do x+1 MM. (Ex. If I need to do a digest for one tube, I will prepare 30µL of MM, but still put 15µL into the tube)
  4. Label the new tubes with corresponding tubes and change the "P" into a "D"
  5. Add 5µL DNA and 15µL MM into each tube
  6. Incubate at 37℃ for at least 30 minutes.
  7. Incubate at 80℃ for 10 minutes to deactivate the enzymes. Wait for digests to cool to room temperature before ligating

How to make gel

  1. Obtain one 125ml conical flask, 1x TAE solution, and agarose
  2. With the balance, measure 0.32g of agarose
  3. Use a measuring cylinder and measure 40 ml of 1x TAE solution
  4. Put the measured powder and liquid into the conical flask (make sure no powder is stuck to the wall)
  5. Use plastic film and wrap around the opening of the flask, BUT remember to leave a hole. Do not completely seal the flask
  6. Microwave the solution for 1 minute
  7. Use a glove and transfer the solution from the Microwave to a black top table
  8. Find the well for the gel, duct tape, and combs (depends on the number of digest you have, you need one or two combs)
  9. Tape the edges of the well and make sure the bottom is sealed. Put the combs in
  10. When the microwaved solution is cooled down a bit, add 4 ul of ethidium bromide into the flask and swirl
  11. Pour the solution into the well and wait til it forms a gel like substance
  12. If you need to store it for future uses, obtain a zip lock bag
  13. Tear off the tape carefully, but leave the combs in
  14. Store them into the refrigerator not the freezer

Run the gel

  1. Put 4µL of dyes into each tube of digest, make sure the dye is well mixed. **NOTE: Only mix dye directly into the digest if using it only for the purposes of running the gel. If the digest is going to be ligated, mix only 10µL of the digest with the dye and run that. This will leave 10µL left over for ligating**
  2. obtain the made gel and put it into the machine (clear one on counter in closet)
  3. Use 1x TAE, and pour it until the gel is totally submerged into the solution
  4. Record which well you are putting the digests into so they are not mixed up
  5. For each row there are eight empty wells. The first place of each row should be the ladder (4µL)
  6. Obtain the total 24µL solution from each tube and add into the gel. **Again, if you are going to be running the digest in a ligation, use only the 14µL prepared earlier (digest+dye) and leave 10µL in the tube**
  7. Close the lid and turn the machine on "high" for approximately 25 minutes
  8. Turn off the machine before removing the lid
  9. Turn off the light and place the gel on the UV light
  10. Turn on the UV light and take a picture, avoiding direct eye contact if possible


  1. Prepare the mix
    • Upstream Part digestion - 2µL
    • Downstream Part digestion - 2µL (replace with water if no Downstream is available, same amount)
    • Destination Plasmid digestion - 2µL
    • 10X T4 DNA Ligase Buffer - 2µL
    • T4 DNA Ligase - 1µL
    • Distilled H2O - 6µL
  2. Put 3µL dH20 in ligation tubes separate of the master mix, and put 2µL of the correct digest into the respective ligation tubes. Make sure to label your tubes well
  3. Put 15µL of the master mix into each ligation tube
  4. Incubate at room temperature for 10 mins
  5. Incubate at 80℃ for 10 mins to deactive the T4 Ligase. Cool to room temperature before transforming into competent cells

Making a Plate

  1. Obtain distilled water, agar, and a 500 ml conical flask
  2. According to the proportion on the label of agar, make a 250-500 ml solution
  3. Use a magnetic stirring bar, tilt the flask and push the bar into the flask
  4. Put the flask on the hot plate, and turn on the stirring button to high
  5. Seal the opening with aluminum foil and autoclave it with standard procedure
  6. Either pour plates when it is still liquid and safe to handle immediately after autoclaving or let solidify
  7. When you need to use it, put it on the hot plate and melt it for about 10 mins
  8. Turn on the magnetic stir
  9. ALWAYS keep an eye on the flask. When the agar melts, remove flask from heat
  10. When it cools off slightly (but is still liquid) add the appropriate antibiotic (300 ml of agar needs 300µL antibiotics)
  11. Normally, 300 ml can pour up to 10 plates, so obtain 12 in case
  12. Pour only a thin layer of agar into each plates
  13. Put corresponding color stripes onto the side of the plates


  1. Put everything you want to sterilize in the pressure cooker
  2. Close the lid
  3. Turn the hot plate to its highest setting
  4. When the pressure cooker starts to rattle (approximately 17psi for ours), set a timer for 15 minutes
  5. When done, put it in the hood and let it cool

Modified Competent Cells

  1. Start liquid culture of DH5 α cells (single colony) from plate. Grow overnight
  2. Get_pre-chilled MCF tubes from fridge
  3. Prepare ice-water bucket
  4. Evenly transfer DH5α cells into MCF tubes
  5. Centrifuge for 10 mins @ 6000 rpm (Use the smaller clinical centrifuge)
  6. Prepare bleach container
  7. Decant supernatant into bleach
  8. Add 400λ of ice cold CCMB80 buffer
  9. Resuspend using the pipet until solution is consistent, then pipet for another 5 cycles (in and out)
  10. Add another 400λ of the buffer. Shake gently by inversion
  11. Place in ice bucket for 20 mins
  12. Centrifuge again for 10 mins. (Same instructions as above)
  13. Decant supernatant into bleach
  14. Resuspend in 175λ of buffer. (Same instructions as above)
  15. Place in ice bucket for 20 mins
  16. Label the float and tubes! (Date and initials!)
  17. Use accordingly or store in large freezer. See "transformation procedure" for more info


  1. Make a "Master Mix"
    1. Obtain a sterile microcentrifuge tube and label it "MM"
    2. Obtain PCR tubes from the back cabinet. For each PCR tube(0.2ml), it needs 19.5*(n+1) µl Sterile distilled water, 2.5*(n+1) µl Buffer, 1*(n+1) µl MgCl2, 1*(n+1) µl dNTP, 0.2 µl Taq, 5*(n+1) µl Genomic DNA. n is the number of PCR tubes you are using.
    3. By sequence, add sterile distilled H2O, Buffer, MgCl2, dNTP, GDNA into Master Mix.
  2. Label the PCR tubes with arabic numbers and place them in the ice box from the freezer.
  3. The first PCR tube will be used as a control, to which add 1µl of sterile distilled water.
  4. For the rest of PCR tubes, add 0.5µl Forward Primers and 0.5µl Reverse Primers respectively. Place them back in the ice box.
  5. Turn on the PCR machine. Choose 002 and start to run.
  6. Add 0.2*(n+1) µl Taq polymerase into the Master Mix.
  7. Take the Master Mix and carefully extract 24µl to each PCR tube.
  8. Place the PCR tubes in the PCR machine.

P.S. You need to make sure the PCR tubes are cold when you place them in the machine. The temperature difference matters, because Taq polymerase is active when it's at room temperature

Survival Curve

  1. Start an overnight culture of bacteria to be tested for alcoholic resistance.
  2. Obtain 16 tubes each with 4 mL LB growth medium and label each with the name of the gene the bacteria being tested has and microliters of alcohol to be put in. The numbers on the tubes should be 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, and 45
  3. Put 4µL antibiotic that the bacteria resist into the tube
  4. Put the amount of isobutanol into the tube as the number written on the side of the tube. If the tube is labelled 2, for example, put 2µL of isobutanol into it
  5. Put 4µL bacteria into the tube
  6. Grow the cultures overnight
  7. Pour the cultures into analysis tubes and centrifuge them for 5 minutes at (rpm or g/something
  8. Pour off the growth medium, add 1x PBS and resuspend
  9. Pour solution into cuvette and place in spectrophotometer measuring the absorbance vs time at 600nm and record the results

Every microliter of isobutanol added to the tube increases the molarity of the growth medium by 2.705mM with respect to isobutanol. The change in volume due to the addition of isobutanol is miniscule.