Molecular cloning of recombinant plasmid-DNAs was performed by using the following standard molecular biological methods that were basically described previously (Sambrook et al., 1989).
5.9.1 Polymerase chain reaction (PCR)
To amplify specific DNA-fragments from plasmids or genomic DNA, polymerase chain reactions (PCR) with corresponding primer pairs (synthesized oligonucleotides) and thermostable DNA-polymerases were performed. For analytic PCRs, Taq-DNA-polymerases (“DreamTaq” from Thermo Scientific) were used, while enzymes with proof-reading activity (“Velocity” from Bioline or “Phusion High-Fidelity” from Thermo Scientific) were preferred for usage of the PCR-product in subsequent molecular cloning reactions. The reaction conditions vary for every enzyme and were chosen as specified by the manufacturers. A standard reaction for the DreamTaq DNA-polymerase is shown in Table 12.
Table 12: Standard reaction and PCR program with DreamTaq DNA-polymerase.
The annealing temperature (x) was chosen according to the melting temperature of the primers.
Component Concentration
(50 µl reaction) Step Time Temperature
10x DreamTaq buffer 1x Initial denaturation 3 min 95°C
DreamTaq DNA-polymerase 1.25 U Denaturation 30 sec 95°C
2 mM dNTP-mix 0.2 mM Annealing 30 sec x
10 µM primer forward 1 µM Extension 1 min/kb 72°C
10 µM primer reverse 1 µM Final extension 10 min 72°C
Template DNA 0.5 µl gDNA or
20 ng plasmids
Storage ∞ 4°C
If required, PCR-products were directly purified by using the “peqGOLD Gel Extraction Kit” (Peqlab) according to the manufacturer’s protocol for PCR clean-up.
Otherwise, the samples were subjected to agarose gel electrophoresis.
5.9.2 Agarose gel electrophoresis and DNA extraction
TAE buffer (pH 8.5): 40 mM Tris-acetate 1 mM EDTA
6x DNA loading dye: 10 mM Tris pH 7.6 60 % (v/v) Glycerol 60 mM EDTA
0.03 % (w/v) Bromophenol blue 0.03 % (w/v) Xylene cyanol
For DNA analyses and preparation of DNA-fragments, 1 % (w/v) agarose gels in 1x TAE-buffer were standardly used. To prepare the gel, 1 g agarose was dissolved in 100 ml of 1x TAE buffer by heating in a microwave, cooled down and supple-mented with 4 µl of an ethidium bromide stock solution (10 mg/ml). Afterwards, the agarose solution was poured into a gel tray and a comb was added to form the wells for the samples. Upon polymerization, the DNA samples were mixed with 6x DNA loading dye and were loaded onto the agarose gel together with a DNA size standard (standardly 10 µl of “Lambda DNA/EcoRI+HindIII Marker” from Thermo Scientific).
Subsequently, gel electrophoresis was performed in 1x TAE-buffer at 120 V for 30 to 60 min and the DNA bands were examined with an UV transilluminator.
If required, specific bands were cut from the gel with a scalpel and the DNA was extracted by using the “peqGOLD Gel Extraction Kit” (Peqlab) according to the manufacturer’s instruction. Finally, the DNA was eluted in 30 µl of elution buffer, the concentration was measured with the NanoDrop spectrophotometer and the
extracted DNA was stored at -20°C.
5.9.3 Restriction digest and dephosphorylation of 5’DNA-ends
Restriction digests with type II restriction endonucleases, which cleave DNA at specific palindromic recognition sites, were performed to analyze plasmids or to produce DNA-fragments with compatible ends for cloning approaches. Enzymes and their corresponding buffers and reaction conditions were used, as suggested from the manufacturers. In general for analytic reactions, 1 µg of plasmid-DNA was digested in a total volume of 20 µl for at least 1 h. For preparative approaches, 5 µg of plasmid-DNA was used in a reaction with a total volume of 50 µl overnight.
To avoid the re-ligation of linearized plasmid-DNAs in subsequent ligation reactions, the 5’phosphate ends from plasmid backbones were dephosphorylated. For that, 1 U of FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) was added directly to the reaction upon digestion of 1 µg of plasmid-DNA. Upon incubation at 37°C for 10 min, the enzyme was inactivated by heating at 65°C for 15 min.
5.9.4 Ligation of DNA-fragments
To produce recombinant plasmids, the chosen DNA-fragments with compatible ends were covalently ligated by the T4 DNA ligase, which catalyzes the formation of a phosphodiester bond between the 5’-phosphate and the 3’-hydroxyl end. The reaction was performed, as suggested by the manufacturer. For one reaction (10 µl), 100 ng of plasmid backbone and twofold excess of insert were used and ligated at 16°C overnight.
5.9.5 Gibson Assembly (GA) reaction
During a Gibson Assembly reaction, multiple DNA-fragments with terminal overlapping regions can be covalently joined in an isothermal one-step reaction, which requires the following three enzymatic activities (Gibson et al., 2009). The T5 exonuclease removes nucleotides from the 5’ends of double-stranded DNAs creating single-stranded 3’overhangs. Thus, such complementary DNA sequences are able to anneal, the Phusion DNA-polymerase extends the 3’ends and the Taq DNA ligase covalently ligates the remaining gaps. Finally, recombinant plasmids are produced. The reaction was performed following the description of Gibson et al.
(2009) and Gibson (2011).
GA-master mix: 5 % (v/v) PEG-800 100 mM Tris-HCl pH 7.5 10 mM MgCl2
10 mM DTT
200 µM of each dNTP 1 mM NAD
0.004 U/µl T5 exonuclease
0.025 U/µl Phusion DNA polymerase 4 U/µl Taq DNA ligase
For one Gibson Assembly reaction (20 µl), 100 ng of linearized plasmid backbone and twofold excess of inserts with homolog ends were diluted in 10 µl of deionized, nuclease-free water and mixed with 10 µl of GA-master mix. Afterwards, the sample was incubated at 50°C for 1 h.
5.9.6 Transformation of E. coli cells with plasmid-DNA
The production of chemically ultra-competent E. coli cells was performed, as described by Inoue et al. (1990). For transformation with plasmids, 100 µl of these competent E. coli cells were thawed on ice, 10 µl of the ligation / Gibson assembly reaction or 100-500 ng of plasmid-DNA was added and incubated on ice for 30 min.
Then, the samples were heat-shocked at 42°C for 2 min, immediately supplemented with 1 ml of LB-medium and incubated at 37°C for 45 to 60 min. This recovery phase was avoided, when plasmids carried an ampicillin resistance gene. Afterwards, the cells were pelleted by centrifugation for 5 min at 3 500 x g, plated onto LB-plates containing the corresponding antibiotics and incubated at 37°C overnight.
5.9.7 Extraction of Plasmid-DNA from E. coli cells
For analytic approaches, the plasmid-DNA from saturated 5 ml bacteria cultures was extracted by using the kit “NucleoSpin Plasmid” (Macherey-Nagel), as described in the manufacturer’s protocol. Finally, the DNA was eluted in 50 µl of deionized, nuclease-free water.
To generate large amounts of plasmid-DNA, 100 ml (high copy plasmids) to 200 ml (low copy plasmids) of saturated bacteria cultures were used to isolate their plasmid-DNAs with the kit “NucleoBond PC 100” (Macherey-Nagel) following the protocol of the producer. Subsequently, the DNA concentration was measured with the NanoDrop spectrophotometer and adjusted to 1 µg/µl with deionized, nuclease-free water. All plasmid-DNA was stored at -20°C.
5.9.8 Sequencing of plasmid-DNA
All modified plasmid regions were sequenced to validate the correct DNA sequence.
For that, 0.5 to 1 µg of plasmid-DNA and 5 µM of the sequencing primer were sent to the company LGC Genomics (Berlin/Germany).
5.9.9 Generation of pHK1349 and pHK1380
Plasmid pHK1349 was created by amplification of the RPL11B ORF + 900 bp upstream of the start codon by HK1485 and HK1486 from genomic yeast DNA. The resulting PCR-product and plasmid pHK12 were digested with SacII and XhoI.
Subsequently, the plasmid backbone and the PCR-product with compatible ends were purified and ligated.
To generate plasmid pHK1380, the URA3 marker gene from pHK1323 was changed into the LEU2 gene. For that, the LEU2 gene including promoter and 3’UTR was amplified by HK1623 and HK1624 from plasmid pHK87 and in parallel, pHK1323 was digested with PstI and NsiI. Afterwards, the purified plasmid backbone and PCR-product with homolog ends were combined by performing a Gibson Assembly reaction.