Journal: Scientific reports

Article Title: High-throughput mutagenesis using a two-fragment PCR approach.

doi: 10.1038/s41598-017-07010-4

Figure Lengend Snippet: Figure 1. Comparison of mutagenesis techniques. In the two-fragment approach, the two mutagenesis primers are separated into two PCR reactions and combined with one reverse primer on the opposite side of the vector respectively, while the one-fragment approach relies on two mutagenesis primers in one PCR reaction.

Article Snippet: For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB).

Techniques: Comparison, Mutagenesis, Plasmid Preparation

Journal: Scientific reports

Article Title: High-throughput mutagenesis using a two-fragment PCR approach.

doi: 10.1038/s41598-017-07010-4

Figure Lengend Snippet: Figure 2. Agarose gel electrophoresis analysis of PCR fragments multiplied by Phusion High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.

Article Snippet: For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB).

Techniques: Agarose Gel Electrophoresis, Residue, Mutagenesis

Journal: Scientific reports

Article Title: High-throughput mutagenesis using a two-fragment PCR approach.

doi: 10.1038/s41598-017-07010-4

Figure Lengend Snippet: Figure 3. Overview of the mutagenesis technique. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates.

Article Snippet: For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB).

Techniques: Mutagenesis, Plasmid Preparation, Amplification, Bacteria, Transformation Assay, Sequencing, Purification

Journal: Scientific reports

Article Title: High-throughput mutagenesis using a two-fragment PCR approach.

doi: 10.1038/s41598-017-07010-4

Figure Lengend Snippet: Figure 5. Troubleshooting scheme. For mutants which were not obtained in the first round of cloning, another clone can be sequenced, if it exists. Furthermore, the Gibson assembly reaction can be redone with the same purified DNA fragments, followed by bacterial transformation and sequencing. For missing mutants, PCR conditions can be changed or PCR containing both mutagenesis primers (one-fragment approach) can be applied.

Article Snippet: For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB).

Techniques: Cloning, Purification, Electroporation Bacterial Transformation, Sequencing, Mutagenesis