Team:CO Mines/Experiments

Miniprep Protocols

Zyppy Plasmid Miniprep Kit from Zymo Research

Monarch Plasmid Miniprep Kit from New England BioLabs

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Store Plasmid Neutralization Buffer at 4°C after opening, as it contains RNase A.

  1. Pellet 1.5 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
  2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer. Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
  3. Lyse cells by adding 200 μl Plasmid Lysis Buffer. Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
  4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer. Gently invert tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
  5. Clarify the lysate by spinning for 2 minutes.
  6. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through. Centrifuge for 1 minute.
  7. Add 400 μl of Plasmid Wash Buffer and centrifuge for 1 minute.
  8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
  9. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Making Lysogeny Broth (LB) medium

Liquid LB (for 1 L)

Ingredients:

  • 10 g Tryptone
  • 5 g Yeast Extract
  • 10 g NaCl

Steps:

  1. Weigh ingredients out into an Erlenmeyer flask.
  2. Add DI water to 1 mL and swirl flask to mix.
  3. Cover the top of the flask with aluminum foil and autoclave tape.
  4. Autoclave for 20 minutes with a slow exhaust.
  5. Allow LB to cool to 55 °C before adding any antibiotics.

LB-agar plates

Follow the same steps as above, but add 15 g of Agar before adding DI water.

Once any necessary antibiotics have been added, pour a thin layer of LB-agar into Petri dishes. If any bubbles form, they can be removed by holding a flame over them.

LB Agar Plates Protocols for Bacteria from Addgene

Restriction Digestion

  1. Select restriction enzymes to digest your plasmid.
  2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
  3. In a 1.5mL tube combine the following:
    • DNA (~1 µL)
    • Restriction Enzyme(s)
    • Buffer
    • BSA (if recommended by manufacturer)
    • nuclease free H2O (nfH2O) up to total volume
      • A typical restriction digestion reaction could look like this:
      • 1 µg DNA
      • 1 µL of each Restriction Enzyme
      • 3 µL 10x Buffer
      • 3 µL 10x BSA (if recommended)
      • x µL nfH2O (to bring total volume to 30-50 µL)
  4. Mix gently by pipetting.
  5. Incubate tube at the manufacturer described temperature and time.
  6. Use gel electrophoresis to confirm if the digestion was successful.

T4 Ligation Protocol

    The reaction is performed on ice. The reaction is scaled to the specifications of the Thermo Fisher T4 DNA Ligase Instructions. The ratio of Insert to Vector was Modified for some ligations.

  1. In a 1.5mL tube combine the following:
    • Vector and Insert DNA
    • T4 Ligase
    • Buffer
    • nuclease free H2O (nfH2O) up to total volume
      • A typical ligation reaction could look like this:
      • 50 ng Vector DNA
      • 37.5 ng Insert DNA
      • 2 µL
      • 2 µL 10x T4 DNA Ligation Buffer
      • x µL nfH2O (to bring total volume to 20 µL)
  2. Mix gently by pipetting.
  3. Incubate tube at the 16°C overnight or at room temperature for 2 hours.
  4. Use gel electrophoresis to confirm if the ligation was successful.

PCR reaction

PCR Protocols for Taq DNA Polymerase from New England BioLabs

For a 25 µL reaction:

  • 2.5 µL of 10x Taq master mix
  • 0.5 µL of 10 mM dNTPs
  • 0.5 µL of 10 uM forward primer
  • 0.5 µL of 10 uM reverse primer
  • 1 µg of template DNA
  • Top to 25 µL with nuclease free water
Thermocycling conditions for a routine PCR:
  • Denaturing: 95 °C for 30 seconds
  • Cycle: Denature for 30 seconds at 95 °C, Anneal for 30 seconds at temperature, Extend for 1 min/kb at 68 °C
  • Hold at 4 °C

Gel electrophoresis

(For a 1 kb gel)
  1. Measure out 0.5 g of agarose and add to an Erlenmeyer flask.
  2. Measure out 50 mL of 1x TAE buffer and add to the flask.
  3. Swirl to mix agarose into the TAE and microwave until agarose is fully dissolved, checking to ensure the mixture does not boil over.
  4. Add 2 µl of ethidium bromide and swirl to mix.
  5. Pour mixture into a gel tray and add gel comb. Allow the gel to sit until solid, about 30 minutes.
  6. Once the gel is solidified, remove the comb and move tray into electrophoresis unit and add 1X TAE until electrophoresis unit is filled.
  7. Add 1 µL of loading dye to 5 µL of DNA.
  8. Add 6 µL of DNA ladder to first gel lane and 6 µL of each labeled sample DNA to the rest of the lanes.
  9. Plug the electrophoresis unit in and run at 120 V for around 40 minutes.
  10. Turn the power off and view gel under ultraviolet light.

DNA Transformation into E.coli

Bacterial Transformation Protocols from Addgene

  1. Allow competent cells to thaw on ice and take out selecting plates to warm up.
  2. Add 2 µL of DNA to 25 µL of competent cells.
  3. Incubate the competent cells on ice for 30 minutes.
  4. Heat shock the cells by incubating them at 42 °C for 30 seconds and then immediately return them to ice for 2 minutes.
  5. Add 500 µL of LB to the cells and place in shaking incubator at 250 rpm and 37 °C for 1 hour. Place plates into a 37 °C incubator to warm up.
  6. Take a sterile loop and plate cells, then return the plates to the incubator overnight.
  7. Check the plates for colonies after incubation.

TOPO TA Cloning

TOPO PCR Protocols from Thermo Fisher Scientific

  1. Add 1 µL of PCR DNA with 1 µL of Topo cloning vector.
  2. Incubate the mixture at room temperature for 5 minutes.
  3. Perform a transformation of competent cells using the Topo mixture and plate.

Kinetic Scan Procedure

  1. All colonies are grown overnight in 5 mL of LB and the respective antibiotic.
  2. The next day a 1/10th dilution is performed and then the OD600 is measured and used to make seperate 0.1 OD600 colonies.
  3. The 0.1 colonies are incubated at 37°C for 1 hour.
  4. The OD600 is measured.
  5. The well plate is set up by placing 100 uL of growth media into each well being used. A set of serial dilutions is then performed using growth media with Cd+2 to achieve the desired Cd+2 concentration.
  6. 100 uL of the colonies are placed into each respective well plate.
  7. The 96 well plate reader is then set to a kinetic scan for 5 hours reading both OD600 and fluorescence. The fluorescence reading is set to 490 excitation and 510 emission. The rotation is set to 275 cpm and the temperature to 37°C.