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.

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.