Methods

Cloning

Agarose Gel Electrophoresis

Towards PCR and enzymatic digest analyses, agarose gels were prepaired with 0,5x TAE and Agarose concentrations between 0.7 % and 0.9 % (w/v). At 65 °C, 1 µl of 10,000x GelRed was added to the Gel before a one hours polymerisation step. As a marker, 1 µl of GeneRuler 1 kb DNA ladder was loaded. Gel runs were commonly performed with voltage ranges between 80 V and 130 V for 45 minutes.

Plasmid Isolation

To purify plasmid DNA from agar plates, either Roti-Prep Plasmid Mini Kit from Carl Roth or High Pure Plasmid Isolation Kit from Roche were used for Minipreps. For high yields of supercoiled, endotoxin-less DNA, Genomed‘s Jetstar Plasmid Purification MIDI Kit or Genopure plasmid MIDI Kit from Roche were used with 150 mL of transformed E. coli o/n culture. No deviances from the manufacturers‘ guidelines were incorporated by performing the isolation steps.

Gel Extraction

When extracting PCR bands or digested fragments from an agarose gel, the QIAquick Gel extraction Kit from Qiagen was used. Steps were performed in accord with provided user‘s manuals, including three deviances: centrifugation steps were always performed with or with less than 17,900 g, columns were rotated by 180° after washing and last, DNA-binding columns were heated to 50 °C for two minutes before the final elution step.

Gibson Assembly

To purify plasmid DNA we used several kits that were provided by our sponsors. For Minipreps we used Peqlab, Roche, qiagen, bioRon, Genetics, Roth. For Midipreps we used Qiagen, Jetstar, Peqlab. Beforehand o/n cultures of transformed E.coli were prepared. No deviances from the manufacturer's guidelines were incorporated by performing the isolation steps.

Ligation

In order to avoid low ligation efficiencies or undesired byproduct vectors, fragment molarities and lengths were considererd as parameters for calculational schemes. Smaller fragments were mostly added excessively within between 4-fold and 8-fold molar amounts, compared to larger backbone fragments. Volume constraint often arose low Gel Extraction yields of particular fragments, so that total DNA quantities were fixed to values at around 25 ng. 5‘ dephosphorylation of single fragments was scarcely performed for reactions with high proportions of backbone religation, using Antarctic Phosphatase. Typical ligation approaches contained 2 µl 10x T4 Buffer, 1-7 µl of each fragment, 1 µl of T4 Ligase and a water fill-up to 20 µl. A 30-minute incubation step was performed at RT, preceding subsequent transformation.

Oligo Annealing

5 µl of 100 µM both Forward and Reverse primer solutions were diluted in 80 µl of water. 10 µl of NEB Buffer No. 2 were added. Samples were heated up to 95 °C for two minutes, whereupon the heating block was switched off. After a 2-hour step of gradual cooling down to RT, the annealed oligos were stored at -20 °C.

Plasmid Isolation

First step of Gibson Assemblies was the rigidly calculation of molar amounts of the fragments. We afterwards joined the fragments and filled them up to 5 µL. These 5 µL were added to 15 µL ice chilled Gibson-Mastermix (Gibson et al., 2009), containing 5x ISO-Buffer, Taq-Ligase and T5 Exonuclease. Except Phusion Polymerase (Gibson et al., 2009) we used the same amount of Q5 Polymerase for the Mastermix. The samples were immediately heated to 50°C for one hour. Afterwards, the samples were cooled for 3 minutes at room temperature and 3 minutes on ice, before they were transformed into competent E.coli.

Polymerase Chain Reaction (PCR)

In order to amplify different DNA-templates, preferably from plasmids, different PCR approaches were used - with the following component amounts for 50 µl of a total volume. 31.5 µl of water, 10 µl of 5x Q5 Reaction Buffer, 4 µl of 2.5 mM dNTP solution, 1 µl of DNA template (200 ng), 1 µl of 10 µM Forward and Reverse Primer, 1 µl of DMSO and 0.5 µl of Q5 High-Fidelity DNA Polymerase. Apart from Touch-down variants and annealing temperature gradient analyses, thermocycler programs consisted of the guideline annotated below.

Assembly PCR

in case of Gibson Assemblies subsequent to fragment amplification, a reduction of fragment quantities to less than five was soon considered appropriate for more efficient Cloning. Assembly PCRs were established for halving the amounts, by using a consecutive double PCR strategy. Expect for Forward and Reverse Primers, all components of a Standard PCR were joined as mentioned above. A first PCR was done with an annealing temperature derived from the sequences of overlapping template regions. Elongation temperature was estimated by considering the fragment length of the larger DNA template (1 kBP per 30 seconds). Five cycles were performed. 2.5 µl of 10 µM Forward and Reverse Primers, both binding to the new 5‘ of both strands were subsequently added to the first PCR mix. A second PCR was performed, with an elongation temperature calculated on the basis of the assembled fragment‘s size.

Colony PCR

In order to screen for positive bacterial clones after transformation experiments, Colony PCRs with available primers were done as follows. One single plate colony was picked and dissolved in 17.8 µl of water in a PCR tube, subsequently 2.5 µl of 10x Standard Taq Reaction Buffer, 1 µl of 10 µM Forward and Reverse Primer, 1 µl 2.5 mM dNTP solution and 0.125 µl Taq Polymerase were added from a previously prepared Mastermix.

Preparative Enzymatic Digest

Buffers for two or more combinatoral enzymes were selected by NEB Double Digest Finder. In order to gain high fragment concentrations in subsequent Gel Extractions, a total volume of 50 µl was mostly chosen. Typical Preparative Digests constisted of 2-3 µg vector DNA, 5 µl 10x NEB Buffer, 0.5 µl of 100x BSA, 1 µl per enzyme and were filled up to 50µl with water. Digests were commonly performed at 37 °C for two hours.

Transformation

3-4 µl of assembled plasmids, derived from either Gibson Assemblies or classical cloning steps, were added to a 25 µl aliquot of chemically competent Top10 E. coli cells on ice. After an incubation period of 30 minutes, a 42 °C heat shock was performed for exactly 45 seconds. Subsequently, a second incubational step on ice was conducted for 2 minutes. 500 µl of previously autoclaved LB medium were added to each aliquot of transformation mix. Samples were incubated at 37 °C and 300 rpm for one hour. Transformations were finalized with plating of 500 µl of transformed E. coli solution on an Ampicillin or Chloramphenicol containing agar plate. Subsequently, plates were stored at 37 °C over night.

Cell culture

Growing conditions

Cells were cultured in DMEM complemented with 10% FCS, glutamin (2 mM), 100 units/ml Penicillin and 100 ug/ml Streptomycin. Cells were grown in humid conditions at 37°C and 7.5% CO₂.

Cell slpitting

DMEM, DPBS and Trypsin solutions were prewarmed to 37 °C. Old medium was aspirated and cells were washed with 5 ml of DPBS, which was subsequently aspirated as well. After an addition of 1 ml Trypsin, an incubation step at 37 °C was performed - until all cells showed signs of detachment. 5 ml of fresh DMEM were then added to recollect trypsinated cells from the dish. Cells were transferred to a Falcon tube and centrifuged at 900 g for 2 minutes. The DMEM-Trypsin supernatant was removed and the pellet finally resuspended in 6 ml DMEM. Seeding into a new 10 cm, 6-well or 24-well dish was performed and cells stored at 37 °C. Confluency was usually checked the day after.

Cell seeding

For a 24-well plate, 0.5 ml cell suspension was commonly utilized per well. Accordingly, 2 ml were added to 6-wells and 10 ml per 10 cm petridish. The amount of cells depended on the respective experiment, but usually ranged from 65,000 in 24 wells to 300,000 in 6-wells and 1,800,000 per 10 cm dishes.