Team:Lund/Experiments

iGEM Lund 2019

Protocols and Experiments

Protocols

Medium Preparation:

LB Media

 For each 1 L reaction mixture, mix the following:

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

Preparation:

  1. Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
  2. Fill with 1 L of distilled water
  3. Stir until all powder is dissolved

TB Media

 For each 1 L reaction mixture, mix the following:

  • 12 g Peptone
  • 24 g Yeast Extract
  • 4 mL Glycerol
  • 2.3 g KH2PO4
  • 12.5 g K2HPO4

Preparation:

  1. Weigh and later autoclave Potassium salts, glycerol separately and peptone along with yeast extract to avoid precipitation
  2. Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

LB Agar Media

 For each 1 L reaction mixture, mix the following:

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

Preparation:

  1. Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
  2. Fill with 1 L of distilled water
  3. After autoclaving, cool the media and add 100 μg/ml of antibiotic of choice (ampicillin) depending on the application
  4. Pour into sterile Petri dishes

PCR - Amplification of gBlocks

MasterMix

 For each 50 µL reaction mixture, mix the following in this order:

  • 38.25 µL MilliQ water
  • 10 µL HF buffer
  • 0.25 µL of each dNTP
  • 0.25 µL Forward Primer
  • 0.25 µL Reverse Primer
  • 0.5 µL Phusion High Fidelity DNA Polymerase

Divide the MasterMix into 10 PCR tubes and add 0.5 µL Template DNA (total volume of 20 µL in each tube) and arrange them in the thermocycler.

    Thermal Cycler Settings:

    1. Initial denaturation: 98 ℃ for 30 seconds
    2. Denaturation: 98 ℃ for 10 seconds
    3. Annealing: 67.4 ℃ (for Trx 61.1 ℃) for 20 seconds (15 cycles)
    4. Extension: 72 ℃ for 20 seconds
    5. Final extension: 72 ℃ for 7minutes
    6. Hold: 4 ℃ until loading

    Gel Electrophoresis

    Gel preparation:

    1. Prepare 2 % agarose gel for gel electrophoresis (0.8 grams agarose powder in 40 mL TAE buffer)
    2. Heat the mixture in the microwave until the solution is clear.
    3. Add 2 µL GelRed after the solution has cooled down.
    4. Fix comb and add the solution into the casket for the gel to solidify.

    Ladder & samples:

    1. Add 2 µL of 100 bp ladder (GeneRuler).
    2. Mix 10 µL of PCR samples with 2 µL loading dye.

    Electrophoresis conditions:

    1. Remove the comb and move the solidified gel to the electrophoresis set up.
    2. Fill the chamber with TAE buffer until all the wells are covered completely.
    3. Load the gel and run it for 90 minutes at 100 V (NOTE: start at 80 V until the samples in the wells move into the gel and then increase to 100 V).

    Colony PCR

    Template preparation and Master Plate

    1. Divide an Agar-plate into four parts using a marker pen.
    2. Inoculate one colony from the transformed TG1 onto one part of the agar plate. Make sure to spread the bacteria within the marked area, and watch out for droplets of water which may spread the bacteria around the plate.
    3. Directly after spreading the bacteria on the plate, dip the inoculation loop in a prepared PCR tube to inoculate this as well.
    4. Incubate plates at 37 ℃ for a few hours or until a suitable amount of growth is seen.
    5. While waiting, continue with Colony PCR.
    6. Inoculate positive transformants from the masterplate in 10ml tubes and incubate at 37 ℃ over night. This is for Sanger Sequencing.

    PCR reaction mixture

    For each 20 µL reaction mixture, mix the following:

    • 2 µL dNTP mix
    • 2 µL Forward primer
    • 2 µL Reverse primer
    • 0.1 µL Taq DNA Polymerase
    • 2 µL Taq buffer
    • 3 µL MgCl2
    • 12 µL MilliQ water

    Thermal Cycler Settings:

    1. Initial denaturation: 95 ℃ for 180 seconds
    2. Denaturation: 95 ℃ for 30 seconds
    3. Annealing: 60 ℃ for 30 seconds (35 cycles)
    4. Extension: 72 ℃ for 20 seconds
    5. Final extension: 72 ℃ for 10 minutes
    6. Hold: 4 ℃ until loading

    Transformations

     For TG1 and BL21-DE3 cells (Heat Shock):

    1. Thaw the competent cells on ice.
    2. Add 10 μL of ligation mixture to 200 μL of cells on ice (maximum concentration of plasmid allowed is 100 ng/mL).
    3. Swirl gently to mix the contents.
    4. Incubate on ice for 1 hour.
    5. Transfer tubes to a heat block with a temperature of 42 ℃ for 45 seconds.
    6. Place samples back on ice and let them cool for 5 minutes.
    7. Add 400 μL of LB media without antibiotics.
    8. Incubate for 45 min in the shaking incubator at 37°C, for the cells to revive.
    9. Centrifuge the tubes in a tabletop centrifuge for 1 minute and remove medium so that only 50 μL is left. Resuspend the cells in the 50 μL that are left.
    10. Plate 50 μL of the culture on LB agar with ampicillin antibiotic and incubate overnight at 37 °C.

    SDS-PAGE

    Reagents:

    1. Make 50 mM sodium acetate buffer at pH 5.0 
    2. Pre-casted gel (Bio-Rad) 
    3. Loading buffer (sample buffer):
      1. 3.3 ml of TRIS 0.5 M pH 6.8 
      2. 8 ml of SDS 10% w/v 25 
      3. 4 ml of glycerol 
      4. 1 ml of bromophenol blue 0.2% w/v 
      5. 2.62 g of DTT (dithiotreithol) 
      6. Adjust volume to 20 ml using MilliQ-water 
    4. Electrophoresis (Running) buffer:
      1. 7.5 g of Tris 
      2. 36 g of glycine 
      3. 2.5 g of SDS 
      4. Adjust pH to 8.3 using HCl and make the final volume up to 500 ml using MilliQ-water. Dilute the buffer 5 times
    5. Molecular weight marker
    6. Staining solution:
      1. 1 g of Coomassie blue 0.2% 
      2. 200 ml of MeOH 40% 
      3. 50 ml of HAc 10% 
      4. 260 ml of MilliQ-water 
    7. De-staining solution:
      1. 40% methanol
      2. 10% acetic acid
      3. 50% MilliQ-water

    Sample Preparation:

    1. Check OD and normalize all the samples to OD 2. 
    2. Resuspend cell pellet in 20 µl of 2x SDS loading dye. 
    3. Boil samples at 100°C for 10 min. 
    4. Load the prepared samples into the gel (Bio Rad plates, 15 well of 15μL, 4 - 20% gradient gel).
    5. After loading, fill the apparatus with the buffer solution between the glass plate until it reaches the maximum volume.

    Running the Gel:

    1. Run the gel at 100 V until the dye front migrates into the running gel (~10 min).
    2. Increase to 150 V until the dye front reaches the bottom of the gel (~30 min). 

    Staining & Destaining the Gel:

    1. Remove the gel from the apparatus. 
    2. Remove the spacers and glass plates, by using the metal spatula. 
    3. Place the gel in a plastic container. 
    4.  Add ~20 ml staining solution (50% methanol, 10% HAc, 0.1% Coomassie Brilliant Blue) and stain for 1 hour on a shaking plate. 
    5. Pour off the staining solution back to the Schott bottle.
    6.  Add ~5 mL destaining solution (50% Methanol, 10% HAc) and destain briefly  for ~1 min.
    7.  Pour off  the destaining solution in the active carbon containing Schott bottle. 
    8. Add ~ 10 mL of destaining solution. 
    9. Destain in a shaking plate until the gel is visibly destained (overnight). 
    10. Pour off  the destain solution in the active carbon containing Schott bottle. 
    11. Rinse with ddH2O. 

    Plamid Isolation

     The protocol according to Nucleospin plasmid kit was followed for Isolation of plasmid with low copy number.

    DNA Purification

    The protocol according to Nucleospin Gel and PCR clean-up kit was followed for DNA purification.

    Restriction Enzyme Cloning

    1. Premix the following in an eppendorf tube:
      • 11 µL MilliQ water
      • 2 µL FD buffer
      • 5 µL Plasmid DNA
    2. Add 1 µL Nde I, mix gently and spin down.
    3. Incubate for 60 min at 37 ℃.
    4. Add 1 µL BamH I, mix gently and spin down.
    5. Incubate for 7 min at 37 ℃.
    6. Incubate for 5 min at 80 ℃ to inactivate enzymes.
    7. 7Add 1 µL of FastAP and incubate for 10 min at 37 ℃.
    8. Incubate for 5 min at 75 ℃ to inactivate enzyme.
    9. Store on ice or at -20 ℃.

    PCR product digestion

    1. Combine the reagents in the following order at room temperature
      • 16 μL MilliQ water
      • 2 μL FastDigest buffer
      • 10 μL DNA (~0,2 μg)
    2. Mix gently and spin down
    3. Add 1 μL Nde1 and incubate at 37°C for 2 hours.
    4. 4Add 1 μL BamH1 and incubate for 10 minutes

    Ligation

    1. Prepare the reaction mixture:
      • 1 : 4 molar ratio (plasmid : gblock)
      • 2 µL Ligase Buffer
      • 1 µL T4 DNA ligase
      • MilliQ water to 20 µL
    2. Incubate for at least 2 hours at room temperature. 
    3. Use 10 uL of ligation mixture in the transformation of 200 uL bacteria.

    Cultivation

  • Incolulate transformed BL21 in 5ml LB media with antibiotic and incubate at 37 °C overnight.
  • Put the samples on ice and measure optical density of the incubated BL21
  • Pipette 0.5ml of culture with OD = 3 at 600nm into the flask containing 100 mL TB media with antibiotic
  • Incubate at 37 °C for 3-4 hours at 160rpm (Check the OD at fixed intervals)
  • Take control samples for SDS-page
  • When OD at 600nm ≈ 1, add 1mM IPTG
  • Grow for 24h and take samples for SDS-page after 2, 4, 8, 16 and 24 hours
  • Pour culture into tubes, centrifuge and save cell pellets