Polymerase Chain Reaction - for Construction

New England Biolabs Phusion High-Fidelity DNA Polymerase

Composition:

Thermocycling Conditions:

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Kapa Biosystems Hifi Hotstart Ready Mix

Thermocycling Conditions:

Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.

Polymerase Chain Reaction - Colony PCR

For screening a large number of clones, single colonies were resuspended in 20 µl LB medium of which 1 µl was used as template for the PCR.

Solis BioDyne FirePol DNA Polymerase

Composition:

Thermocycling Conditions:

New England Biolabs Taq DNA Polymerase

Composition:

Thermocycling Conditions:

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Site-Directed Mutagenesis (QuickChange)

Thermocycling Conditions:

Primers were designed according to the guidlines of The Richard Lab ¹

• The targeted mutation should be included into both primers.
• The mutation can be as close as 4 bases from the 5'-terminus.
• The mutation should be at least 8 bases from the 3'-terminus.
• At least eight non-overlapping bases should be introduced at the 3-end of each primer.
• At least one G or C should be at the end of each primer.
• Design your primers (including the mutations) to have a Tm >=78°C.

The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. In order to obtain high base exchange efficiency, the template need to be removed from the reaction thoroughly.

Preparation of Competent Cells

Preparation of Chemically Competent Cells

• Inoculate 100 ml of prewarmed LB medium with 1 ml overnight culture and grow the bacteria to an OD₆₀₀ of 0.5.
• Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4°C)
• Carfully discard supernatant, keep cells always on ice.
• Resuspend cells in 30 ml cold TFB1 and incubate on ice for 90 minutes.
• Centrifuge for 5 min (4000xg, 4°C)
• Carfully discard supernatant, keep cells always on ice.
• Resuspend the cells in 4 ml ice-cold TFB2 buffer.
• Prepare aliquots of 100 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Chemically Competent Cells

• Thaw the competent cells on ice
• Use 50 ul of the competent cells for one transformation, do not add more than 5 ul of DNA to the cells (10% of the volume of the competent cells)
• Incubate them for 20 min on ice
• Incubate them for 90 s at 42° C and add afterwards 500 µl of SOC
• Let them regenerate for 1 h at 37° C with shaking
• Plate a part of the culture according to your expectations on LB-agar plates with the approbriate antibiotic.

Preparation of Electrocompetent Cells

• Inoculate 600 ml of prewarmed LB medium with 6 ml overnight culture and grow the bacteria to an OD₆₀₀ of 0.4.
• Cool the culture on ice fpr 30 min, transfer 300 ml of the culture into 50 ml tubes and centrifuge them for 10 min (3000xg, 4°C). Discard supernatant and add the rest of the culture. Again discard supernatant.
• Carfully resuspend cells in ice-cold, autoclaved and deionized water and fill up to 50 ml
• Centrifuge again for 10 min (3000xg, 4°C) and repeat wash step from above
• Carfully resuspend cells in ice-cold 10% glycerol (working always on ice)
• Centrifuge for 10 min (3000xg, 4°C)
• Discard supernatant
• Resuspend the cells in the remaining glycerol solution.
• Prepare aliquots of 35 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Electrocompetent Cells

• Thaw the electrocompetent cells on ice. Pre-chill also the electroporation cuvette.
• Add DNA to the cells. Attention: the DNA should contain as little ions as possible. Purify or dilute reactions containing salt.
• Transfer the bacteria/DNA mix into a pre-chilled electroporation cuvette and electroporate them.
• Add IMMEDIATELY 500 µl of SOC and transfer them back into the microcentrifuge tube.
• Let them regenerate for 1 h at 37° C with sufficient shaking.
• Plate a part of the transformation mix according to your expectations on LB-agar plates with the approbriate antibiotic.

Plate reader experiment

• Day -1: Restreak

  Restreak all experiments you want to run from cryostocks

• Day 0: Prepare precultures

  Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.

• Day 1

  • Prepare and divide into PCR tubes all PBS amounts necessary to make the dilutions of DETA/NO from the stock. Place PCR tubes in a row next to each other. This eases work with multichannel pipette. Use “experimental calculations” excel sheet to calculate DETA/NO dilutions.
  • Open Tecan iController. Open "Heating" on settings and click ON to set temperature to37 degrees before the experiment starts.

  • OD600 measurement and dilution

    • Measure OD600 using the spectrometer. Place 1 mL of liquid (overnight culture) in the cuvette, make sure the arrow on the cuvette aligns with the arrow on the machine. Also don’t touch the lower parts of the cuvette as it can interfere with the measurement.
    • Blank spectrometer with M9. If it is not possible to measure OD, dilute the culture 1:1 and try again.
    • Dilute preculture to OD=0.1 with M9 and add antibiotics. Take a single packaged sterile 96-well plate. Pipette 200 uL per well and seal completely without touching the seal. Place the plate in the plate reader. Keep in mind the plate reader OD is 1/4th of the actual OD.
    • Dilute precultures and let them shake in the plate reader for at least 2 hours before induction to make sure cells are in the exponential phase during induction. Add a blank with M9 media in the well plate.

  • Setting up the experiment

    Design

    • Design experiment such that you have triplicates of each sample to be tested.
    • On Tecan iController, select the type of plate you are using. The standard one is Greiner 96 Flat Transparent.
    • Set the parameters of the Tecan plate reader protocol:
      • Temperature: 37 degrees.
      • Shaking: 10 seconds, Amplitude 6, Mode: orbital.
      • Kinetic Cycle: pick 100 cycles
      • Set OD measurement to 600. Set number of flashes to 25.
      • add shaking step for 10 seconds with amplitude 6
      • add Fluorescence Intensity. For GFP set excitation to 488nm and emission toemission 530nm. The numbers that appear next to nm is the +/- range of the excitation/emission. So pay attention that the range of excitation and emission you get never overlaps, or the measurements will not be accurate. Number of flashes:25. Settle time:0ms. Mode: Top. Z-position: Manual, 20000um. Lag time 0us. Integration time 20us. Label: Green Fluorescence.
      • add additional shaking step of 900 seconds with amplitude 6mm and orbital mode and hit start.
      As soon as you out the plate in the plate reader, prepare serial dilutions of DETA/no in PBS.

• Measurements:

References:

• [1] Tom Richard, Department of Agricultural and Biological Engineering, Penn State University.
• [2] Gibson, Daniel G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature methods 6.5 (2009): 343-345.