Gel extraction kit

Manufactured by Bio Basic

Sourced in Canada

The Gel Extraction Kit is a laboratory tool designed to extract and purify DNA fragments from agarose gels following electrophoresis. It provides a reliable and efficient method to isolate specific DNA bands of interest for downstream applications such as cloning, sequencing, or further analysis.

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9 protocols using gel extraction kit

Cloning and Mutagenesis of Arabidopsis Immunity Genes

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The coding sequences of Arabidopsis thaliana NPR1 (AT1G64280), NPR4 (AT4G19660) and NIMIN2 (AT3G25882) were amplified by polymerase chain reaction from an Arabidopsis cDNA library, and sub-cloned into the DH5α vector with different N-terminally fused tags and a tobacco etch virus (TEV)-cleavage site. The specific amino acid changes for the NPR1 and NPR4 point mutations were generated using the QuikChange II site-directed mutagenesis kit (Agilent). Glutathione-S-transferase (GST)-fused NPR4 coding sequence (CDS) was subcloned into the pFastBac vector and transformed to E. coli DH10Bac for making baculovirus for protein expression in insect cells. Protein domain swaps were generated by amplifying the desired regions of each CDS with primers designed to create overlapping sequences for each fragment. The DNA fragments were amplified in separate PCR reactions, processed with either a PCR clean-up kit or gel extraction kit (Bio Basic Inc.), and the desired fragments were fused by PCR using gene-specific forward and reverse primers containing attB1 and attB2 Gateway™ recombination sequences, respectively. All CDSs were recombined into pDONR207 or pDONR221 and subsequent expression vectors using the Gateway™ technology and sequenced to confirm accuracy.

Wang W., Withers J., Li H., Zwack P.J., Rusnac D.V., Shi H., Liu L., Yan S., Hinds T.R., Guttman M., Dong X, & Zheng N. (2020). Structural Basis of Salicylic Acid Perception by Arabidopsis NPR Proteins. Nature, 586(7828), 311-316.

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Cloning and Mutagenesis of Arabidopsis Immunity Genes

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Genomic DNA Extraction and Plasmid Manipulation

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Primers used in this study are shown in Table S1. Genomic DNA was isolated using the 5′ ArchivePure DNA kit (5 Prime, Gaithersburg, MD). PCR products and plasmids were purified using a commercially available gel extraction kit (Bio Basic Inc., Markham, Ontario) and a plasmid miniprep kit (Fermentas, Glen Burnie, MD) respectively. Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs (Beverly, MA). Standard methods for the manipulation of plasmids and DNA fragments were followed (Sambrook et al., 1989) . Sequences of all cloned PCR products were verified at the University of California Davis Sequencing Facility using fluorescent automated DNA sequencing with an Applied Biosystems 3730 automated sequencer.
Escherichia coli strains were transformed with plasmid DNA (Sambrook et al., 1989) , and plasmids were mated into P. putida strains by conjugation in the presence of E. coli HB101(pRK2013) (Simon et al., 1983) . Exconjugants were selected on MSB plates containing 10 mM succinate and the appropriate antibiotic. Deletion mutants that arose from double-crossover events were isolated by counterselection in MSB containing 10 mM succinate and 20% sucrose. Mutants were screened for antibiotic sensitivity to confirm the loss of plasmid, and deletions were verified by PCR using the appropriate primers (Table S1).

Luu R.A., Kootstra J.D., Nesteryuk V., Brunton C.N., Parales J.V., Ditty J.L, & Parales R.E. (2015). Integration of chemotaxis, transport and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY. Molecular microbiology, 96(1).

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Glycoprotein J Gene Amplification and Phylogenetic Analysis

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Glycoprotein J (gJ) gene was amplified from virus isolated from SPF eggs using gJ gene specific primers (Craig et al., 2017) . PCR reaction was carried out with Q5HiFi Taq DNA polymerase (NEB ® ) with GC enhancer. PCR mixture was kept in a PCR thermal cycler with the following conditions: 98 °C -30s; 35 cycles of 98 °C -30s, 62 °C-45s and 72 °C for 90s, final extension at 72 °C for 10 minutes. Bulk PCR products were purified with Biobasic ® gel extraction kit and sent for Sanger dideoxy sequencing to a commercial sequence service provider. Sequence was aligned using BioEdit™ software and aligned sequence was submitted to GenBank (Accession number -MT997150.1). phylogenetic analysis was carried out with MEGA[X]™ software using Tamura-Nei model, maximum likelihood method with 1000 bootstrap values (Tamura and Nei (2013) ; Kumar et al., 2018) .

Jaisree S. (2021). Detection and Molecular Characterization of Avian Infectious Laryngotracheitis Virus Isolated from a Breeder Flock. Journal of animal research, 11(2).

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DNA Extraction from Agarose Gel

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Agarose gel containing the relevant DNA band was excised and the excess of agarose was excised to minimize the gel slice weight. 300mg of the slice was transferred into a microcentrifuge tube for DNA extraction with the gel extraction kit (Bio Basic Inc. ). The eluted pure DNA products were stored at -20°C for future use.

Haque S. (2021). Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7.

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Macrolide Biosensor Development in E. coli

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The strains and plasmids used in this study are listed in Table S1. E. coli strains 10G (Lucigen) and TOP10 (Invitrogen) were used for cloning and biosensor expression, respectively. Aeromicrobium erythreum NRRL B3381 and the corresponding knock-out strain were a kind gift from Prof. Eric Miller, NC State University. The plasmid pMLGFP was used as a template for MphR mutagenesis and to express the reporter gene in the biosensor strains. Bacteria were grown in Luria Broth supplemented with ampicillin and tetracycline as appropriate. ErA, clarithromycin, azithromycin, and roxithromycin were from Sigma-Aldrich and pikromycin was from Abcam. YC-17 was a kind gift from Prof. David Sherman, University of Michigan. Each macrolide was prepared in dimethyl sulfoxide (DMSO) to a stock concentration of 50 mM, 5 mM, 500 μM, or 50 μM. All other chemicals were purchased from Sigma-Aldrich unless stated otherwise. PCR products were extracted with a Bio Basic Gel Extraction Kit. Restriction enzymes were purchased from New England Biolabs. Plasmids were isolated using a plasmid miniprep kit from Bio Basic.

Kasey C.M., Zerrad M., Li Y., Cropp T.A, & Williams G.J. (2017). Development of transcription factor-based designer macrolide biosensors for metabolic engineering and synthetic biology. ACS synthetic biology, 7(1), 227-239.

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Bacterial Protein Expression and Purification

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All other chemicals were purchased from Sigma-Aldrich unless stated otherwise. PCR products were extracted with a Bio Basic Gel Extraction Kit. Restriction enzymes were purchased from New England Biolabs. Plasmids were isolated using a plasmid miniprep kit from Bio Basic. Other DNA preparation kits (genomic, and gel extraction) were purchased from (NEB). Oligos were synthesized by Integrated DNA Technologies (IDT) and purified by IDT using standard desalting. Polymerases were purchased from Fisher Scientific; all restriction enzymes were purchased from New England Biolabs (NEB). LB media was purchased from Fisher Scientific. Polyacrylamide gels were homemade and prepared using reagents purchased from Fisher Scientific; gels were prepared using 4% acrylamide stacking gel and 20% acrylamide running gel. Dibenzocyclooctyne-fluor (DBCO) 488 reagent was purchased from Sigma Aldrich. Reagents used for buffer preparation were purchased from VWR. Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from CalBioChem. Bacillus thuringiensis 4BD1 strain was ordered from the Bacillus Genetic Stock Center (BGSC). For protein expression, E. coli strain BL21(DE3) was used unless stated otherwise. For DNA storage and manipulation, E. coli TOP10 (Invitrogen) was used.

Carpenter S.M, & Williams G.J. (2018). Extender Unit Promiscuity and Orthogonal Protein Interactions of an Aminomalonyl-ACP Utilizing Trans-Acyltransferase from Zwittermicin Biosynthesis. ACS chemical biology, 13(12), 3361-3373.

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Macrolide Antibiotic Biosensors in E. coli

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The strains and plasmids used in this study are listed in Supplementary Table S2. E. coli strains 10G (Lucigen) TOP10 (Invitrogen) were used for cloning and biosensor expression, respectively. Bacteria were grown in Luria Broth (Fisher Scientific) supplemented with ampicillin (Fisher Scientific) and tetracycline (Fisher Scientific) as appropriate. ErA and CLA were obtained from Sigma Aldrich. Each macrolide was prepared in dimethyl sulfoxide (DMSO) to a stock concentration of 0.5 μM, 5 μM, 50 μM, or 500 μM. DMSO and 96-deepwell plates were purchased from Fisher Scientific. All other chemicals were purchased from Sigma-Aldrich unless stated otherwise. PCR products were extracted with a Bio Basic Gel Extraction Kit. All enzymes for DNA manipulations were purchased from New England Biolabs. Plasmids were isolated using a plasmid miniprep kit from Bio Basic. All oligonucleotides were purchased from Integrated DNA Technologies. Clear and opaque flat-bottom 96-well plates were purchased from Greiner Bio-One. All other chemicals were reagent grade or better.

Li Y., Reed M., Wright H.T., Cropp T.A, & Williams G.J. (2021). Development of a genetically-encoded biosensor for reporting the methyltransferase-dependent biosynthesis of semi-synthetic macrolide antibiotics. ACS synthetic biology, 10(10), 2520-2531.

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Cloning and Localization of CLEC19A

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All of the fragments were amplified by Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, USA), according to the manufacturer's instructions. For the cloning of the related fragments, the PCR products were purified using the BioBasic gel extraction kit (BioBasic, Canada), and cloned into pCDNA3.1( +) or pEGFP-N1 vectors. The full-length cDNA of the CLEC19A gene was amplified from the cDNA of the NTERA-2 (NT2) cell line and cloned into the pCDNA3.1( +) vector (Addgene) between the sites of EcoRI (Thermo Fisher Scientific, USA) and NotI (NEB, USA) (pCL19A) (Fig. S2A). To determine the localization of the CLEC19A gene, its ORF sequence without stop codon was fused to the EGFP sequence of a pEGFP-N1 vector (Addgene). Briefly, the ORF sequence was amplified from the cDNA of the NT2 cell line and inserted between the EcoRI (Thermo Fisher Scientific, USA) and SalI (NEB, USA) sites of the pEGFP-N1 vector in a frame with the EGFP sequence (pCL19A-ORF) (Fig. S2B). The sequences of all plasmid constructs were confirmed by colony PCR [42 ] and sanger sequencing [43 (link)].
The cells were transiently transfected with vectors used in this study using Lipofectamine ® 3000 (Thermo Fisher Scientific, USA). The cells were seeded into a plate 24 h before transfection. Next, the plasmid DNA-lipid complex was prepared according to the manufacturer's protocol and was added to cells.

Mohajerani F., Tehrankhah Z.M., Rahmani S., Afsordeh N., Shafiee S., Pourgholami M.H., Soltani B.M, & Sadeghizadeh M. (2024). CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme. BMC Cancer, 24, 19.

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