All strains were grown in lysogeny broth (LB), Rich Defined Media (RDM, Teknova) or on LB agar, supplemented with 50 μg ml−1 kanamycin, 100 μg ml−1 carbenicillin or 25 μg ml−1 chloramphenicol (Sigma) for selection. Plasmids were isolated using a QIAQuick Miniprep kit (Qiagen). Polymerase chain reaction (PCR) reaction products were purified using a GeneJet Gel extraction kit (Thermo Scientific) or NucleoSpin Gel and PCR Clean-Up kit (ClonTech). Plasmids and PCR products were sequenced using Sanger sequencing (Elim Biopharma or MCLab). All PCR and cloning reactions were performed on a S1000 Thermal Cycler (Bio-Rad). Information about the E. coli strains used in this experiment can be found in the Supplementary Data .
Qiaquick miniprep kit
Manufactured by Qiagen
The QIAQuick Miniprep kit is a product designed for the rapid and efficient purification of plasmid DNA from bacterial cultures. It utilizes a silica-membrane technology to bind plasmid DNA, which is then eluted in a small volume of buffer.
Automatically generated - may contain errors
Lab products found in correlation
Carbenicillin, by Teknova (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up kit, by Takara Bio (1 mentions)
S1000 thermal cycler, by Bio-Rad (1 mentions)
Sanger sequencing, by Eton Bioscience (1 mentions)
Qubit fluorometric quantitation, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sybr master mix, by Roche (1 mentions)
5 protocols using qiaquick miniprep kit
1
Bacterial Strain Cultivation and Plasmid Isolation
2
Molecular cloning techniques in Pseudomonas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All plasmids generated and/or used in this study are listed in Table S1 . Routine DNA manipulations including extraction, restriction, ligation, electroporation, conjugation and agarose gel electrophoresis were performed using standard molecular methods (Sambrook and Russell, 2001 ). Plasmid extraction was completed using a Qiagen™ QiaQuick miniprep kit following the manufacturer's instructions. The TcR marker of pMINI-CTX1 derived constructs integrated into the chromosome of P. aeruginosa was removed using the Flp recombinase system as previously described (Hoang et al., 1998 (link), 2000 (link)). All primers used for DNA amplification by PCR are detailed in Table S2 . DNA sequencing was conducted at the University of Nottingham's DNA sequencing facility.
3
Validating Sensitivity and Specificity of RMC Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the sensitivity and specificity of the RMC assay in our hands we generated a PCR product which contained a mtDNA mutation in the RMC site and one which was wild-type in the RMC site. A pCR-scriptTM Amp SK(+) cloning Kit (Stratagene) was used to clone the products following the manufacturer's protocol. Recombinant plasmids were identified by blue–white colour selection and the cultures grown up using a Qiaquick miniprep kit (Qiagen). The DNA was then extracted and quantified and the wild-type and mutant PCR products mixed at concentrations ranging from 100% wild-type to 100% mutant. The RMC assay was then carried out as above and the observed mutant fractions calculated and compared to the expected fractions (Table S4 ). There was no difference between observed and expected fractions, confirming the RMC assay to be both highly sensitive and specific.
4
CRISPR-Cas9 Genome Engineering in Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CRISPR-Cas9 genome engineering was performed on the S. cerevisiae ABC16-Monster strain using vectors p414 and p426 obtained from the Church lab (Addgene) as previously described29 (link). To produce gRNA plasmids specific to the desired mutation sites, oligonucleotides were synthesized (Integrated DNA Technologies) to match the target sequence and contain a 24 base-pair overlap with the p426 vector backbone. Gene-specific gRNAs were amplified by PCR, transformed into StellarTM competent E. coli cells (Takara) and selected on LB-Ampicillin plates. DNA was isolated from transformed E. coli cells and purified using the QiaQuick Miniprep kit (Qiagen) and quantified via Qubit Fluorometric Quantitation (ThermoFisher). Cas9-expressing ABC16-Monster cells were transformed with 300–500 ng of gene-specific gRNA vector and 1–2 nmole of synthesized donor template (IDT) containing the desired base-pair substitution via standard lithium acetate method. Transformed cells were selected on methionine and leucine deficient CM-glucose plates. Each mutation was confirmed with Sanger sequencing (Eton Bioscience). Sequences of primers and oligonucleotides used for this study can be found in Supplementary Table 3 . Edited yeast strains generated for this study are listed in Supplementary Table 2 .
5
qRT-PCR: Sensitive RNA Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qRT-PCR was performed as described before (Onishi et al., 2012 (link)). Briefly, RNA was isolated using the QIAquick Miniprep kit (QIAGEN) and reverse transcribed with Superscript III (Life Technologies). PCR was performed on the ABI 7000 using SYBR Master mix (Roche).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!