Kod hot start dna polymerase

gpt1/alg6 S. pombe (Sp61G4A (h-, ade6-M210, ade1, leu1-32, ura4-D18, gpt1::ura4-D1684, alg6::ura4+) was used for heterologous expression (Fanchiotti et al. 1998 (link)). S. pombe cells were grown at 28° in YEA medium or MM medium supplemented with adenine or leucine as needed (Moreno et al. 1991 (link)). Escherichia coli strain STBL3 (Invitrogen, Carlsbad, CA) was grown in LB medium with 100 µg/ml ampicillin when needed. Reagents for yeast media were obtained from Difco Laboratories (Detroit, MI). N-Methyl-1-deoxynojirimycin (NMDNJ) was from Research Chemicals (North York, ON, Canada). Enzymes used for DNA procedures were from New England Biolabs (Ipswich, MA), KOD Hot Start DNA Polymerase was from Merck (Darmstadt, Alemania) andpCR2.1-TOPO Vector was from Invitrogen (Carlsbad, CA). Unless otherwise stated, all other reagents were from Sigma (St. Louis, MO). UDP-[¹⁴C]Glc was synthesized as previously reported with slight modifications (Wright A 1965). Protein concentrations were determined by Bio-Rad Protein Assay as described by the manufacturer.

Mutant yeast stains (listed in Supplementary Table 1) were engineered as described previously^{24 (link)}. Briefly, the PrimerQuest tool from IDT (Integrated DNA Technologies) was used to design PCR primers (see Supplementary Table 2 for the list of primers used), and DNA fragments were generated using KOD Hot Start DNA polymerase (EMD Millipore). Yeast transformation was performed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research), followed by a two-step selection method using either synthetic media with uracil-dropout amino acid mix (SC/URA) or 5-fluorouracil (5-FOA) as the selective agents. All newly engineered and mutated yeast strains were confirmed by PCR and sequencing.

Wild-type and 2-FA-resistant colonies were analyzed by colony PCR with Chelex-100 resin (Bio-Rad) following a previously reported method [47 ]. Briefly, a fraction of the colony was added to a 200 µL tube which contained 50 µL of a 5% Chelex-100 solution. Tubes were vigorously agitated and then briefly centrifuged and incubated for 20 min at 98 °C. One microliter of this solution was used per 50 µL PCR reaction. To amplify the 1132-bp PCR product, which contains the target site for both sgRNAS, primers APT-Fw (5′-CTTATTCACAAGGTCGAATC-3′) and NVDF278 (5′-TTGTGACGTTACACACTGCCTC-3′) were used (Supplementary Figure S1). PCR was carried out using KOD Hot Start DNA polymerase (Merck Millipore, Burlington, MA, USA) following the manufacturer’s instructions and using the following cycling conditions: 3 min at 94 °C, then 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, followed by a final extension of 5 min at 72 °C. The PCR products were analyzed by agarose gel electrophoresis (1%) stained with Gel Red (Biotium Inc., Hayward, CA, USA), bands around 1 kb were excised and purified with Wizard SV Gel and PCR Clean-Up System (Promega, Fitchburg, WI, USA).

mito-QC mice were generated as previously described [19]. Autophagy reporter mice (mCherry-GFP-MAP1LC3B, referred to as mCherry-GFP-LC3) were generated in the same way as mito-QC using targeted transgenesis by TaconicArtemis GmbH as described [20]. Mice were maintained on a C57BL6/J background. Genotyping was performed by diagnostic end-point PCR using genomic DNA isolated from tissue biopsy specimens. WT and KO alleles were detected using KOD Hot Start DNA polymerase (EMD Millipore, 71086) and manufacturer-recommended conditions. All animal studies and breeding were approved by the University of Dundee ethical review committee, and further subjected to approved study plans by the Named Veterinary Surgeon and Compliance Officer (Dr. Ngaire Dennison) and performed under a UK Home Office project license in accordance with the Animal Scientific Procedures Act of 1986.

Isolation of E. faecalis genomic DNA was performed using a ZymoBIOMICS DNA Miniprep Kit (Zymo Research). All PCR used for cloning were performed with high fidelity KOD Hot Start DNA Polymerase (EMD Millipore). E. faecalis ΔessB was generated by allelic replacement by cloning an in frame essB deletion product into pLT06 using Gibson Assembly Master Mix (New England Biolabs), integrating this construct into the chromosome, and resolving the deletion mutant by homologous recombination [105 (link)–107 (link)]. For ectopic expression of putative immunity proteins, coding regions of OG1RF_11110, OG1RF_11112, OG1RF_11122, and OG1RF_12413 were cloned downstream of the bacA promoter (P_bacA) by restriction digestion and ligation into the shuttle vector pLZ12A [20 (link)]. Coding regions of ireK and OG1RF_11099 were cloned downstream of the cCF10 responsive promoter (P_Q) by restriction digestion and ligation into pCIE and pCIEtm vectors, respectively. As attempts to clone OG1RF_11121 by itself were unsuccessful, we cloned the OG1RF_11121 and OG1RF_11122 open reading frames, which overlap by 13 base pairs, together under the P_Q promoter in pCIEtm plasmid. Primer sequences and restriction enzymes used for cloning are listed in S1 Table. Plasmids were introduced into electrocompetent E. faecalis cells as previously described [20 (link)].

In vitro transcription, capping and polyadenylation of ARMeD mRNAs was performed using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher AM1345) and purified with the MEGAclear Transcription Clean-Up Kit (Thermo Fisher AM1908) according to the manufacturers’ instructions. DNA templates were PCR amplified from the corresponding plasmids using the KOD Hot Start DNA Polymerase (Merck Millipore 71086) and purified by agarose gel electrophoresis using the QIAquick MinElute Gel Extraction Kit (QIAGEN 28604). The used primer sequences are shown in Table S3.