RNA isolation from in vitro cultivated axenic metacestode vesicles and primary cells was performed using a Trizol (5Prime, Hamburg, Germany)-based method as previously described [8 (link)]. For reverse transcription, 2 μg total RNA was used for cDNA synthesis using oligonucleotide CD3-RT (5’-ATC TCT TGA AAG GAT CCT GCA GGT26 V -3’). PCR products were cloned using the PCR cloning Kit (QIAGEN, Hilden, Germany) or the TOPO XL cloning Kit (Invitrogen). The complete list of primer sequences used for emmekk1, emgrb2, emmkk3, emmkk4, emmkk5, and emmpk3 cDNA amplification and characterization is given in S1 Table . Identification of genes of interest is discussed in conjunction with results below. Upon cloning, PCR products were directly sequenced using primers binding to vector sequences adjacent to the multiple cloning site by Sanger Sequencing (Microsynth Seqlab, Göttingen, Germany). The sequences of all genes newly characterized in this work have been submitted to the GenBank, EMBL, and DDJB databases under accession numbers listed in S1 Table .
Sanger sequencing
Manufactured by Microsynth
Sourced in Switzerland, Germany
Sanger sequencing is a DNA sequencing method used to determine the nucleotide sequence of a DNA sample. It is a widely used technique for small-scale DNA sequencing projects.
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Lab products found in correlation
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (4 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (3 mentions)
Gibson assembly, by New England Biolabs (2 mentions)
Nebuilder hifi dna assembly cloning kit, by New England Biolabs (2 mentions)
Dpni, by New England Biolabs (2 mentions)
Genejet gel extraction and dna cleanup micro kit, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Pcr cloning kit, by Qiagen (1 mentions)
Topo xl cloning kit, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
Pwo dna polymerase, by Roche (1 mentions)
Taq dna polymerase gotaq, by Promega (1 mentions)
63 protocols using sanger sequencing
1
Axenic Metacestode Vesicle RNA Isolation
2
Heterologous Expression of Methanol Dehydrogenase and Hexose Metabolism Enzymes
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Primers and plasmids used in this study are listed in Supplementary Data 4 . The heterologously introduced plasmids were pSEVA424 with the methanol dehydrogenase 2 (mdh) variant CT4-145 (link) from Cupriavidus necator and pSEVA131 with the 3-hexulose 6-phosphate synthase (hps) and the 6-phospho 3-hexuloisomerase (phi) from Methylobacillus flagellatus29 (link). The nucleotide sequence of the plasmids was confirmed by PCR and Sanger sequencing (Microsynth AG; Switzerland). Plasmid maps are available from the Source Data file.
3
Validation of ADAR1-Mediated Transcript Editing
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Genomic DNA (gDNA) and RNA were prepared from RC-K8 cells, including ADAR1 WT and KO. gDNA was extracted using the High Pure PCR template Preparation kit (Roche) following manufacturer instructions. PCR amplification was then performed using Q5 High-Fidelity DNA Polymerase (NEB). RNA was extracted using the RNeasy Mini kit (Qiagen) and treated with DNase (Invitrogen). A one-step RT-PCR kit (#210212, QIAGEN) was used. The PCR products were purified (#740609, Macherey-Nagel) and analyzed by Sanger sequencing (Microsynth AG, Switzerland). Quantification of editing was performed directly from the chromatogram using MultiEditR.89 (link) The editing sites identified in DLBCL patients within CARD11 and TP73 transcripts by RED-ML were validated by amplifying the targeted region from both genomic DNA and RNA followed by cloning (#K1231, ThermoFisher) and Sanger sequencing.
4
Cloning and Sequence Verification
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Standard restriction digestion and ligation was used for cloning of the expression and reporter vectors used in this study. Dual-luminescence or dual-fluorescent vectors contain a heptameric nucleotide sequence between the two proteins encoded by the construct, causing a +1 frameshift to be required for translation of the second protein. Sanger sequencing (Microsynth AG, Switzerland) was used to verify the sequences of all constructs.
5
APOE Genotyping and Sex Differences
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Data on age and sex (self-reported as women or men) were ascertained at the clinical visit. APOE genotyping was performed by commercially available Sanger Sequencing (Microsynth AG). Participants were dichotomized into individuals carrying at least one copy of the ε4 allele and APOE4 non-carriers.
6
Directed Mutagenesis of AncOR5_75 Receptor
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Individual mutations were introduced in the pCS2+/AncOR5_75 plasmid using the Q5 Site-directed Mutagenesis kit (New England Biolabs, Ipswich, MA, USA) with primers designed using NEBaseChanger (https://nebasechanger.neb.com/ , SI Appendix, Table S3 ). Double and triple mutation combinations were selected based on the 3D structure analysis as residues that are in close proximity to each other. Plasmids were checked for successful mutations by Sanger sequencing (MicroSynth AG, Balgach, Switzerland) and then mini-prepped using QIAprep Spin Miniprep kit (Qiagen). For the AncOR5_75 receptor carrying eight mutations (AncOR5_75_mut8x), the ORF was synthesized in vitro by Synbio Technologies and subcloned into the pCS2+ vector.
7
Cloning and Mutagenesis of CPB Constructs
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The codon optimized ORF (Genscript) encoding CPB was cloned as N‐terminal His6 or N‐terminal GST fusion into pET19‐b (Novagen) using NcoI and BamHI sites. CPB containing a C‐terminal His6‐tag was generated using Q5® site‐directed mutagenesis kit (NEB), pET‐19b_His6_CPB as template and primer pair #1 and elongation time of 4 min to delete the N‐terminal His6, followed by a second PCR step using primer pair #2 and elongation time of 4 min to introduce the C‐terminal His6. The His6_CPBΔ23 construct was generated using Gibson Assembly® (NEB) and pET‐19b_His6_CPB was linearized using NcoI and BamHI sites. A PCR product containing His6‐CPBΔ23 was generated with primer pair #4, Ta and an elongation time of 1 min. CPB chimera constructs were generated using Gibson Assembly® master mix (NEB) and pET‐19b_His6_CPB was linearized using NcoI and BamHI sites. PCR products containing the codon optimized N‐terminal domains of δ‐toxin (His6‐CPBΔ23δ‐toxin(1–24)), Hla (His6‐CPBΔ23Hla(1–20)) and HlgB (His6‐CPBΔ23HlgB(1–19)) were generated using pET‐19b_His6_CPB as template and indicated primer pairs, Ta and an elongation time of 1 min (Appendix Fig S1 ). The resulting sequences were verified by Sanger Sequencing (Microsynth AG) and plasmids were transformed into E. coli Dh5α competent cells (NEB) for amplification.
8
Plasmid Construction for CRISPR/Cas9 Genome Editing
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Briefly, SpCas9-gRNA expressing plasmids (targeting human and mouse genes and also containing TL1 and TL2 spacers) were generated from the px330 vector (Addgene # 422303 (link)), and cloning spacers into pmCherry_gRNA plasmid was conducted according to Hanhui Ma et al.47 (link) The construction of pmCherry_gRNA, pX330-Flag-ehSpCas9 and pX330-Flag-wtSpCas9 is described in (Kulcsár et al. 2017 submitted). The nuclease inactive SpCas9 (dCas9) expressing plasmid was created from px335 (Addgene #423353 (link)) by using Body Double cloning method.48 (link) The self-cleaving plasmids were created by using Gibson assembly method. The self-cleaving plasmid containing a puromycin cassette (pSc1-puro) was constructed by using pLKO.1puro plasmid (Addgene #845349 (link)). The self-cleaving plasmid with a degradation domain (pSc1-DD) was created from pBMN DHFR(DD)-YFP plasmid (Addgene #2932550 (link)). The Prnp gene targeting homologous recombination donor plasmid was created by using nested PCR. The Rosa26 locus homologous recombination donor plasmid was created by using pDonor-MCS-rosa26 plasmid [Addgene #3720051 (link)]. For more detailed information see Supplementary Materials and Methods . The sequences of all plasmid constructs were confirmed by Sanger sequencing (Microsynth AG).
9
Standard Molecular Biology Protocols
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Recombinant DNA techniques were performed following standard molecular–biology-based protocols (Sambrook et al., 1982 ). DNA-modifying enzymes such as Pwo DNA polymerase (Roche), Taq DNA polymerase (GoTaq; Promega), and restriction modification enzymes (New England Biolabs) were used according to the manufacturer’s recommendations. Genetically modified strains were verified by colony PCR and, if required, also by Sanger sequencing (Microsynth, Switzerland) for their correctness.
10
Alu Region PCR and Sanger Sequencing
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The localization of editing sites was retrieved from REDIportal [23 (link)], which contains editing sites from different databases: RADAR [24 (link)], DARNED [25 (link)], and Atlas [26 (link)]. We considered only sites present in at least two databases. For PCR of different Alu regions, cDNA was used. PCR was performed in a total volume of 50 μL containing 1× Green Go TaqR Flexi Buffer (Promega), 2 mM MgCl2 solution, 0.2 mM PCR Nucleotide Mix, 0.2 mM of each primer, 1.25 U of Go TaqR G2 Hot Start Polymerase (Promega), and 25 ng of cDNA. The primers used are listed in Supplementary Table S1 . Products were confirmed by electrophoresis on a 2% agarose gel and excised. After purification according to the Macherey-Nagel NucleoSpin® Gel and PCR clean-up protocol, products were sent for Sanger sequencing (performed by Microsynth AG, Balgach, Switzerland). Raw sequencing outputs were quantified with ImageJ software.
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