Team:UANL Mty-Mexico/Notebook/protocols

From 2012.igem.org

iGEM UANL 2012


Protocols

  • Preparation of E. coli Calcium Chloride competent cells
      1. Inoculate a single colony into 5 mL of LB media without any antibiotics and grow overnight at 37 °C with vigorous shaking.
      2. Inoculate 1 mL of the desired strain into 100 mL of fresh LB, use a 500 mL flask.
      3. Incubate at 37 °C with vigorous shaking until 0.3 - 0.4 sideOD600
      4. Put the flask on ice. Pre-chill 50 mL centrifuge tubes and the centrifuge itself at 4°C.
      5. Centrifuge 50 mL of the culture at 8,000 rpm for 5 minutes at 4 °C.
      6. Remove the supernatant and add 10 mL of cold CaCl2 0.1 M. Vortex until the pellet is resuspended.
      7. Incubate on ice for 30 minutes, shake the tube once in a while.
      8. Centrifuge at 8,000 rpm for 5 minutes at 4°C. Remove the supernatant and add 2 mL of CaCl2 0.1 M. Resuspend carefully using a micropipette. Keep always on ice.
      9. Mix the two preparations in a tube and store on ice, or use for transformation.

      Note: The competent cells can be stored on ice up to two weeks.


  • Preparation of E. coli Rubidium Chloride competent cells
      1. Inoculate 5 mL of LB broth with DH5α and incubate the culture overnight at 37°C with vigorous shacking.
      2. Use small culture to inoculate 100 mL of LB broth. Incubate at 37°C with shacking until the culture reaches an optical density (OD595) 0.4-0.6.
      3. Transfer to two 50 mL centrifuge tubes.
      4. Spin at maximum speed for 5 minutes at 4°C.
      5. Remove supernatant.
      6. Add 20 mL of TBF1 and resuspend the pellet.
      7. Incubate on ice for 20 minutes.
      8. Pour off supernatant.
      9. Centrifuge 5 minutes at 8000 rpm/4°C.
      10. Resuspend cellular pellet with 4 mL of TBF2.
      11. Place aliquots of 100 µL at -70°C.
  • Heat-shock transformation of E. coli competent cells
      1. Add 50 µL of Ca+2 competent cells to a pre-chilled centrifuge tube. Keep always on ice until step 4.
      2. Add plasmid DNA (100 ng) or ligation (up to 5 µL) depending on DNA concentration.
      3. Use 1 µL of a 1 ng/µL DNA sample as positive test in a separate tube. It is recommended to use a DNA-free negative test tube as well.
      4. Chill the tube on ice for 20 - 30 minutes.
      5. Expose the reaction mixture to a 42ºC 1 minute heat-shock.
      6. Put the tube on ice for 2 minutes.
      7. Add 200 µL of antibiotic-free LB media.
      8. Incubate at 37ºC for 20 - 30 minutes.
      9. Spread the appropriate quantity of cells (50-200 µL) on selective LB agar plates.
      10. Incubate overnight at 37º C.
      11. The positive plate must have around 1,000 colonies as an optimal (1X106 transformants per µg supercoiled DNA).

      Notes: Until heat-shock, handle the tubes from the upper part to avoid warming the cells. Low temperature is critical for successful transformation. Avoid transforming with more than 5 µL of ligation mixture, as ligation buffer may reduce transformation efficiency.


  • Preparation of Electrocompetent E. coli cells
      1. Inoculate a single colony of E. coli in 5 mL of LB media. Grow overnight or for 5 hours at 37°C with shaking at 250 rpm.
      2. Inoculate 2.5 mL of the previous culture in 200 mL of LB media in a 2 L flask. Grow at 37 °C shaking at 300 rpm until the culture reaches an OD of 0.5 - 0.7.
      3. Chill the cells on ice for 10 - 15 minutes and then transfer the cells into a pre-chilled centrifuge bottle.
      4. Centrifuge at 4,200 rpm for 10 minutes at 2 °C (Beckman J-6M).
      5. Remove the supernatant and resuspend the pellet in 5 mL of cold water. Add 200 mL of cold water and mix well. Centrifuge at 4,200 rpm for 10 minutes at 2 °C.
      6. Remove the supernatant and resuspend the pellet by shaking gently in the remaining liquid volume.
      7. Add 200 mL of cold water, mix well and centrifuge at 4,200 rpm for 20 minutes at 2°C.
      8. Add 20 mL of 10% cold glycerol and mix well. Centrifuge at 4,200 rpm for 20 minutes at 2 °C.
      9. Add 10 mL of 10% cold glycerol to each tube. Resuspend and gather all the content of the tubes in a single tube, centrifuge and remove the supernatant.
      10. Estimate the pellet volume and add an equal volume of 10% cold glycerol. Resuspend the cells.
      11. Divide the final volume into pre chilled tubes (100 μl) and store at -80 °C.

      Note: Pre-chill all the materials that will be in contact with the cells.


  • Electroporation of E. coli competent cells
      1. Take a tube with 50 µL of electrocompetent E. coli cells, thaw on ice.
      2. Add a volume containing 100 ng of DNA.
      3. Carefully transfer the cell/DNA mix into a pre-chilled electroporation cuvette. Make sure to deposit the cells at the bottom and not to introduce any air bubbles.
      4. Electroporate under the following conditions:
      5. Immediately add 250 µL of SOC media to the cuvette.
      6. Incubate with vigorous shaking (250 rpm) at 37 °C for 1 hour.
      7. Add 750 µL of LB media and mix by pipetting up and down.
      8. Spread 200 µL of cells onto a selective LB agar plate.

      Note: All must be performed on ice. Electroporation cuvettes are previously chilled on ice. DNA and bacteria must be thawed on ice too.


  • Mini preparation of plasmid DNA
      1. Pour 1.5 mL of the culture in a 1.5 mL microcentrifuge tube and centrifuge at 14,000 rpm for 30 seconds. Remove carefully the supernatant.
      2. Add 200 µL of Solution I. Resuspend the pellet by using vortex briefly or by pipetting up and down. Incubate at room temperature for 5 minutes.
      3. Add 200 µL of Solution II and mix gently by inverting and rotating the tube several times. Do not vortex. Incubate at room temperature for 5 minutes.
      4. Add 200 µL of Solution III and mix gently by inverting and rotating the tube several times. Incubate the tube on ice for 5 minutes.
      5. Centrifuge at 14,000 rpm for 5 minutes.
      6. Transfer the supernatant to a fresh tube containing 1 mL of 100% ethanol.
      7. Incubate at -20 ºC for 10 minutes. (Max. 2 h)
      8. Centrifuge at 14,000 rpm for 10 minutes. Remove the supernatant.
      9. Add 200 µL of 70% ethanol and vortex gently for 10 seconds.
      10. Centrifuge at 14,000 rpm for 5 minutes. Remove the supernatant by pipetting. Aspirate off any residual supernatant.
      11. Dry at 37ºC for 5 minutes.
      12. Add 20 µL of H2O + 20 µg/mL of RNase. Resuspend by using vortex briefly.
      13. Run an agarose gel (0.8%) or store at 4 ºC.
  • Midi preparation of plasmid DNA (AXYPREP ™ PLASMID MIDIPREP KIT)
      1. Collect 30 of overnight LB culture for the preparation of the high-copy plasmid, or 100 mL of overnight LB culture for the preparation of the low copy-plasmid. Centrifuge at 3000X G for 8 minutes to pellet the bacteria. Discard the supernatant. Invert the centrifuge tube on a paper towel for 1 minute to drain off residual medium.
      2. Resuspend bacterial pellet with 4.5 mL of Buffer S1.

        a. Make sure that RNase A has been added into Buffer S1.

        b. Incompletely resuspension will reduce lysis efficiency, plasmid yield and purity.

      3. Add 4.5 mL of Buffer S2, and mix by gently inverting tube 6-8 times (do NOT vortex)

        a. Buffer S3K must be added within 5 minutes.

        b. Check the Buffer S2 for precipitation before each use. If precipitation occurs, incubate at 37°C.

      4. Add 4.5 mL Buffer S3K pre-chilled at 4°C, invert the tube 10 times. Incubate at room temperature for 5 minutes.
      5. Add 4.5 mL of Buffer B (pre-chilled at 4°C), gently invert the tube 10 times.
      6. Centrifuge at 6000 G for 10 minutes.
      7. Attach the AxyPrep Vacuum Manifold base to a vacuum pump. Position the manifold top with luer-type fittings on the manifold base. Attach a Midiprep Plasmid Column to a complimentary fitting on the manifold top. Make sure that the column is firmly seated.
      8. Transfer supernatant from step 5 to the Midiprep Syringe Filter. Carefully insert the plunger into the syringe filter and push with a steady motion. Discharged the filtrate into the Midiprep Plasmid Column.
      9. Switch on the vacuum source and continue to apply vacuum to the column until lysate remains. Do not turn the vacuum off.
      10. Keep vacuum, add 7 ml of Buffer W1 and draw the solution through the Midiprep Plasmid Column.
      11. Add 8 mL of Buffer W2, and draw through the Midiprep Plasmid Column.

        a. Make sure that ethanol has been added into Buffer W2 concentrate

      12. Use the plastic wrench to detach the end component from the Midiprep Plasmid Column assembly and place it into a 1.5 mL microfuge tube. Add 300 µL of Buffer W2 and centrifuge at 12 000 G for 2 minutes.
      13. Transfer the end component to a fresh tube 1.5 mL microfuge tube. Add 300 µL of distilled water to the center of the membrane. Let it stand at room temperature for 1 minute and centrifuge at 12 000 G for 1 minute to elute the plasmid DNA.
      14. Note: This protocol was taken from AxyPrep Midi and Maxi Plasmid Kits, available at http://static3.jadedpixel.com/s/files/1/0001/7488/files/axyprep_midi_maxi_plasmid_protocol.pdf

  • Solutions for Mini preparation of Plasmid DNA

      Solution I (200 mL)

      milliliters or grams

      - Tris HCl 1 M (pH 8.0)

      5 mL

      - EDTA 0.5 M (pH 8.0)

      4 mL

      - Distilled H2O

      Bring the final volume up to 200 mL


      Solution II (200 mL)


      - NaOH 10N

      4 mL

      - SDS (powder)

      2.0 gr

      - Bidistilled H20

      Bring the final volume up to 200 mL


      Soll III (200 mL)


      - Potassium acetate (CH3CO2K)

      58.8 gr

      - Acetic acid (CH3-COOH)

      23.0 mL

      - Distilled H20

      Bring the final volume up to 200 mL


      Notes: When preparing Solution II, first add a little bidistilled water, then add NaOH and dissolve carefully SDS. Finally, bring the final volume up to 200 mL with bidistilled water.

      When preparing Solution III, first add 100 mL of H2O and then the potassium acetate. Once it has been add the acetic acid and finally bring the final volume up to 200 ml with bidistilled water.


  • Agarose Gel Electrophoresis Protocol

      To be Loaded:


      • DNA molecular size marker (λ PstI): 2 - 3 μL

      • Plasmid DNA: 2 - 3 μL

      • Enzyme restrictions: 10 μL

      • PCR products: 5 μL


      Procedure:

      1. Prepare an agarose gel of the desired concentration (see Agarose gels section).
      2. Add the necessary SB 1X buffer into the electrophoresis tank to cover the gel.
      3. Load the first well with marker, and then load the DNA samples mixed with loading buffer into the wells.
      4. Plug in the anode and cathode cables so that the DNA samples can move through the gel toward the anode.
      5. Run the electrophoresis at 200 volts.
      6. Wait approximately 20 - 30 minutes or until the bromophenol blue reaches the end of the gel and stop the electrophoresis.

      Note: DNA moves toward the positive electric field (anode, usually red cable) due to the negative charges.


  • Agarose gel

      Concentration for supercoiled and plasmid DNA: 0.8%

      For digestion reaction fragments over 1,000 bp: 0.8%

      For digestion reaction fragments below 500 bp: 1.5%

      DNA size marker (λ + PstI): Use 2 or 3 μL per gel.

      Note: Not needed when running supercoiled DNA samples, like plasmid DNA.


  • SB buffer 20X

      SB (Sodium Borate) electrophoresis buffer, 20X Stock:

      1. In 700 mL of distilled H2O, dissolve 8 gr of NaOH.
      2. Weight 51 of Boric Acid and dissolve ¾ parts in the NaOH solution
      3. Dissolve the remaining Boric Acid until the buffer reach pH 8.0.
      4. Complete to 1 L with distilled H2O and store in a sterile flask.

      Note: Use SB 1X as buffer to run agarose gels up to 200 volts


  • Ethidium Bromide Gel Staining

      1. Dilute the stock to 20 μg/mL in a special container with the gel buffer.
      2. Put the gel into the container.
      3. Let it stain for 3 - 5 minutes.
      4. Take the gel out of the container and soak the stained gel in water for 5 minutes or more to clear background ethidium bromide from the gel.
      5. View the gel under a UV light source or on a UV transilluminator.

      Note: If you want to use ethidium bromide, confine its use to a small area of your laboratory. Wear gloves when staining, handle stained gels, and dispose of any waste.


  • Lambda/PstI Molecular Size Marker
      And-gate
      Lambda molecular size marker.

      Mix:


      • phage λ DNA (500 ng/μL) 50.0 μL
      • PstI 2.5 μL
      • Buffer 10X 6.0 μL
      • H20 1.5 μL

      Procedure:

      1. Mix the ingredients listed above
      2. Incubate at 37ºC / 45 minutes
      3. Again add 2.5 μL PstI
      4. Incubate 37ºC / 45 minutes
      5. Add 6.0 μL Loading buffer 6X

      Check on agarose gel.


      Notes: Final concentration: 0.30 μgr/μL. Final Volume: 66.0 μL.


  • Restriction enzyme digestion of DNA

      Mix for 1 reaction, final volume of 20 µL

      Add the following to a microcentrifuge tube:

      DNA

      2-3 µg

      Buffer 10x

      2.0 µL

      Enzyme (10 U/µL)

      0.3 µL (1 enzyme unit per µg DNA)

      H2O

      Until 20.0 µL


      Incubate the mixture at 37 °C (it may change, check enzyme specifications) for 1 - 1.5 hours.

      Note: Prepare a mix when possible to minimize enzyme handling.


  • PCR

      PCR reaction mix:

      DNA template

      Total 100 ng (In 25 μl)

      Buffer 10x

      2.5 μL

      Mg++ 50 mM

      0.75 μL

      dNTPs 20 mM

      0.25 μL

      Primer Fwd 100 ng/μL

      0.50 μL

      Primer Rv 100 ng/μL

      0.50 μL

      Taq Pol 5 U/μL

      0.25 μL

      H2O

      To bring the volume up to 25 μL

      Procedure:

      1. Add the corresponding H2O to a sterile PCR tube.
      2. Add the rest of the components but the enzyme and DNA.
      3. Add the enzyme, mix gently.
      4. Add the respective DNA sample and mix gently.
      5. Spin the tube briefly.
      6. Place the sample in the thermocycler and start your PCR program.

      Notes:

      Put on gloves before taking the PCR mix components out of the freezer.

      DNA must be added at last because it may form complexes with Mg++ and inhibit the reaction.

      When possible, make a mix with all the common components to minimize enzyme waste.


  • Antibiotics

      Antibiotic

      Final concentration

      Stock concentration

      µL per mL

      Spectinomycin (Sp)

      100 µg/mL

      20 µg/µL

      5

      Ampicillin (Amp)

      50 µg/mL

      10 µg/µL

      1

      Kanamycin (Kan)

      50 µg/mL

      50 µg/µL

      1

      Chloramphenicol (Cm)

      34 µg/mL

      34 µg/µL

      1

      Tetracycline (Tet)

      10 µg/mL

      5 µg/ µL

      2


      Notes:

      Always verify stock concentration, in case of unknown assume the one indicated above.

      When using more than one antibiotic simultaneously use half the concentration for each antibiotic.


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