Team:Gaston Day School/Experiment

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Experiments and Protocol

Labels

For MCF tubes:

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

Transformation

  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, Chl for complete, Amp for parts)
  4. Make an ice-water bath
  5. Obtain X MCF tubes and a float
  6. Add 50 ul of competent cells
  7. Add 2ul of the DNA
  8. Place it in the ice-water bath for 2 mins
  9. While waiting, turn on the sink's hot water, obtain a beaker, add hot water, and using a thermometer, adjust water to 42o C (using the hot plate if needed)
  10. Place tubes in the incubator for 30 seconds
  11. Another 2 mins on ice
  12. Add 1 ml of warm SOC medium
  13. Place each tube in the incubator (37℃) for 1 hour
  14. Plating

Pick Colonies

  1. Obtain the green sticks, empty LB tubes, and the plates from the incubator
  2. Label the tubes with corresponding plates
  3. Spot the colonies (Choose more than one if available)
  4. Use the sticks and gently obtain the colonies
  5. Swirl around in the LB solution and dispose the stick into the tip trash
  6. Put the tubes into the shaking incubator 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 75% 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. Place the tubes upside-down and allow them to dry completely
  15. Resuspend the pellet in 25 uL TE
  16. Use 10 uL of the DNA in a restriction digest or 1 uL for PCR

Digest

  1. obtain the plasmid prep tubes (in incubator), new MCF tubes, enzymes in the freezer
  2. For each new tube, prepare the master mix(MM):
    • water 15ul
    • buffer 2ul
    • Upstream enzyme 0.5ul
    • Downstream enzyme 0.5ul
  3. Each tube needs 18ul 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 30ul of MM, but still put 18ul into the tube)
  4. Label the new tubes with corresponding tubes and change the "P" into a "D"
  5. Add 3ul DNA and 15ul MM into each tube
  6. Obtain a tube float and put all the tubes into the dry incubator for at least an hour.

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 4ul of dyes into each tube of digest, make sure the dye is well mixed
  2. obtain the made gel and put it into the machine (clear one on counter in closet)
  3. Use 0.25x TAE, and pour it until the gel is totally submerged into the solution
  4. Take a paper and record the order of tubes when you put them in
  5. For each roll there are eight empty places **The first place of each roll have to be the ladder (4ul)
  6. Obtain the total 24ul solution from each tube and add into the gel
  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 backroom light and place the gel on the UV light
  10. Turn on the UV light and observe

Ligation

  1. Prepare the mix
    • Upstream Part digestion - 2ul
    • Downstream Part digestion - 2ul (replace with water if no Downstream is available, same amount)
    • Destination Plasmid digestion - 2ul
    • 10X T4 DNA Ligase Buffer - 2ul
    • T4 DNA Ligase - 1ul
    • Distilled H2O - 11 ul
  2. Incubate in the incubator for 10 mins
  3. Heat activate at 80oC for 2 mins (hot plate)

Making a Plate

  1. Obtain distilled water, agar, and a 500 ml conical flask (Blue cabinet)
  2. According to the proportion on the label of agar, make a 300 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. Pull it down and let it cool until solidified
  7. When you need to use it, put it on the hot plate and heat it up for about 10 mins
  8. Then, turn on the stirring button
  9. ALWAYS keep an eye on the flask. When all the solid turns into liquid, stop heating the flask
  10. Transfer the flask to a black surface and when it is warm, add appropriate antibiotics in it (300 ml of agar needs 300 ul 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

Autoclave

  1. Put everything you want to sterile in the big pressure cooker
  2. Close the lid
  3. Turn the hot plate to 10
  4. Until you hear the "ziiiii" sound, time for 15 mins
  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

PCR

  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 18.5*(n+1) µl Sterile distilled water, 2.5*(n+1) µl Buffer, 1*(n+1) µl MgCl2, 1*(n+1) µl dNTP, 0.5*(n+1) µl VR, 0.5*(n+1) µl VF2, 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, VR, VF2, 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 tap polymerase is active when it's at room temperature.

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