A 5′- and 3′-terminal-truncated NP of NSDV was amplified as described above and subcloned into pFastBac (Invitrogen) and then used to transfect E. coli DH10Bac (Invitrogen). The resulting recombinant bacmids were purified with the PureLinkHiPure Plasmid Miniprep kit (Invitrogen) and used to transfect sf9 insect cells (Invitrogen) with Cellfectin II reagent (Invitrogen). Following incubation for 4d, recombinant baculovirus particles were harvested and incubated with sf9 cells (Invitrogen) in 96-well plates for 3 days. The IIFA was performed in a similar manner to that of SFTSV described above.
Purelink hipure plasmid miniprep kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PureLink™ HiPure Plasmid Miniprep Kit is a set of reagents and materials designed for the rapid and efficient purification of plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to capture and purify plasmid DNA, which can then be eluted for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellfectin 2 reagent, by Thermo Fisher Scientific (4 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (2 mentions)
Bromophenol blue xylene cyanol based loading dye, by Carl Roth (2 mentions)
Doc print 2, by Vilber (2 mentions)
Bacpak baculovirus rapid titer kit, by Takara Bio (2 mentions)
Pfastbac, by Thermo Fisher Scientific (1 mentions)
Sf9 insect cells, by Thermo Fisher Scientific (1 mentions)
Dh10bac e coli, by Thermo Fisher Scientific (1 mentions)
Sf9 cells, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trizol reagent rnaiso plus, by Takara Bio (1 mentions)
Mda mb 231 cell line, by American Type Culture Collection (1 mentions)
Mir 34a mimic, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Abi prism 3130xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
22 protocols using purelink hipure plasmid miniprep kit
1
Recombinant Baculovirus Production for NSDV
2
Plasmid Transfection Optimization in MDA-MB-231 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM basic medium and OPTI-MEM were from HyClone (Logan, UT, U.S.A.). Fetal bovine serum, trypsin and penicillin–streptomycin solutions were obtained from Life Technologies (Carlsbad, CA, U.S.A.). The MDA-MB-231 cell line was from ATCC (Manassas, VA). The cell counting kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). The plasmid encoding enhanced green fluorescent protein (pEGFP) was kept in our laboratory. The PureLink™ HiPure Plasmid Miniprep kit was purchased from Invitrogen (Carlsbad, U.S.A.). The miR-34a mimics, cy5-labeled miR-34a mimic and miRNA negative control (miR-NC) were synthesized by Genepharma (Shanghai, China). TRIzol reagent (RNAiso Plus) was purchased from Takara (Dalian, China). RevertAid Reverse Transcriptase was purchased from Thermo Fisher (Runcorn, Cheshire, U.K.). Primers were synthesized by Sagon Biotech (Shanghai, China). The 2× SGExcel FastSYBR mixture and miRNA qPCR master mix (SYBR Green) was purchased from Sangon Biotech (Shanghai, China). The Notch1, Hes1 and GAPDH antibodies were from OmnimAbs (CA, U.S.A.). CMBs (Uphere™ Trans+) were obtained from Trust Bio Sonics (Taiwan, China).
3
DNA Cleavage Assay of Copper Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nuclease activity of the compounds was determined using plasmid pRS325II DNA, which was a gift from Steven Haase (Addgene plasmid #35467, 6835 base pairs) [71 (link)]. The plasmid was amplified in Escherichia coli by transforming One Shot® TOP10 chemically competent E. coli (Invitrogen, Thermo Fisher Scientific). The plasmid was isolated from positive colonies using a PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen, Thermo Fisher Scientific). Plasmid (200 ng/sample) was exposed to complexes (1 ) and (2 ) in the presence of 1 mM hydrogen peroxide (to exploit the Cu(II/III) redox couple) or 1 mM ascorbic acid (to exploit the Cu(I/II) redox couple) [46 (link)]. The DNA cleavage experiments were done in a 9/1 (v/v) ratio of 50 mM Tris-HCl, pH 8, and DMSO. The samples were incubated for 1 h at 37 °C before a bromophenol blue/xylene cyanol-based loading dye (Roth, Germany) was added. The samples were loaded onto a 1% (w/v) agarose gel containing 1 μg/mL EtBr in 1 × TBE (Tris–boric acid–EDTA) buffer. Electrophoresis was performed at 50 V for 60 min in 1 × TBE buffer. The images of the fluorescent ethidium bromide-stained gels were captured using a gel documentation system (Doc-Print II, VilberLourmat, France). The cleavage experiments were done three times, with similar results. One representative gel is shown.
4
Amplification and Sequencing of Comammox Nitrospira
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplification of Comammox Nitrospira Clade A and Clade B amoA and nxrB genes was carried out by PCR from gDNA of LURDF soil sample using Platinum®Taq DNA Polymerase, High Fidelity (Invitrogen™ Thermo Fisher Scientific, Waltham, MA, United States) to prevent error propagation, ensuring correct replication of the target DNA. The resultant PCR products were purified and concentrated using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) followed by cloning into pGEM®-T Easy Vector System (Promega Corporation, Madison, WI, United States) and transformed into Escherichia coli JM109 high-efficiency competent cells, according to the manufacturer’s protocols. Plasmid of prominent colonies were extracted using PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen™ Thermo Fisher Scientific, Waltham, MA, United States) and confirmed with restriction digest using EcoRI-HF restriction enzyme and subsequently subjected to Sanger sequencing using T7 and SP6 primers by ABI Prism 3130xl Genetic Analyser (Applied Biosystems™, Waltham, MA, United States). Sequence results were analyzed by MUSCLE alignment (Edgar, 2004 (link)) using Geneious Prime® (Biomatters Ltd., Auckland, New Zealand).
5
Transient Expression of Recombinant Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genes encoding heavy chains and light chain were inserted separately into pcDNA3.4 and amplified in E. coli DH5α. PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen) was used for low endotoxin plasmid preparation. Monoclonal antibodies were transiently expressed by co-transfecting ExpiCHO-S cells (ThermoFisher) with heavy chain and light chain plasmids using an ExpiCHO™ Expression System (Gibco). Cell culture was harvested after an 8–14 day of incubation at 37 °C with humidified atmosphere of 8% CO2 with shaking. Full-length IgG was obtained by affinity purification utilizing a Protein A chromatography column (GE Healthcare) in AKTA avant (Cytiva). For long-term storage, antibodies were kept in a solution containing 10 mM Histidine-HCl, 9% trehalose, and 0.01% polysorbate 80.
6
Cloning and Mutagenesis of Protein-Coding Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein-coding segments were cloned from the cDNA library by conventional PCR. At the 5′ and 3′ termini of each gene, 20 nucleotides were synthesized and used as primers. The second set of primers with sequences of the indicated restriction enzymes was utilized to insert gene segments into plasmid pET-51b (Table 2 ). Mutagenesis PCR was used for inserting coding segments into plasmid pcDNA4 (a gift from Elena Oancea, Brown University). Several fusion tags (HA, His6, MBP, GST, Trx, and mCherry) were also inserted into the plasmids at the indicated positions by mutagenesis PCR. Moreover, single-site mutations were constructed by mutagenesis PCR. Q5 high-fidelity 2× master mix (New England BioLabs [NEB], Ipswich, MA, USA) was used for PCR. A PureLink quick gel extraction and PCR purification combo kit and a PureLink HiPure plasmid miniprep kit (Invitrogen) were used to recover DNA and extract plasmids from E. coli . One Shot TOP10 chemically competent E. coli cells from Invitrogen were utilized for the transformation procedure. The sequences of DNA segments and constructed plasmids were confirmed by sequencing, done by Genewiz (Cambridge, MA, USA).
7
Phagemid Isolation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phagemid DNA was isolated from individual clones which showed specificity for their target antigen. Clones were grown overnight in 2YT-AmpGlu media, then phagemids isolated using Purelink Hipure Plasmid Mini Prep kit (Invitrogen). Phagemid scFv inserts were sequenced by Sanger sequencing at the Australian Genome Research Facility (AGRF, Brisbane Australia), using phagemid-derived forward and reverse primers.
8
Recombinant Baculovirus PD-1 Mutant Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant baculoviruses encoding the PD-1 mutant were constructed using the Bac-to-Bac baculovirus expression system previously described [24 (link)]. Briefly, a pFastBac1 plasmid with the membrane protein sequence of interest was transformed into DH10Bac Escherichia coli, which contained a bacmid and helper plasmid encoding the transposase gene. The target gene inserted between Tn7 transposon sequences was transposed into the bacmid. Colonies containing the recombinant bacmid were identified by blue/white selection and the bacmid was isolated using a PureLink HiPure Plasmid Miniprep Kit (Invitrogen). Sf9 cells were transfected with the recombinant bacmid using Cellfectin II Reagent (Invitrogen) and incubated at 27 °C for 5 days. The supernatant containing P1 viruses was collected, and the viral concentration was amplified three times. The viral titers were determined by a BacPAK Baculovirus Rapid Titer Kit (Takara Bio USA, Inc., San Jose, CA, USA).
9
Plasmid Isolation and DNA Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids were isolated from E. coli with either PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen) or PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen). PCR products and DNA fragments were purified with GFX™ PCR DNA and Gel Band Purification Kit (Cytiva).
The concentration and purity of nucleic acid solutions were estimated with NanoDrop ND‐1000 Spectrophotometer (Thermo Scientific), and their integrity was assessed by agarose gel electrophoresis.
To visualize agarose gel electrophoresed DNA molecules, gels containing GreenSafe premium (NZYTech) nucleic acid stain were imaged with ChemiDoc XRS+ Gel Imaging System (BIORAD). The 1 Kb Plus DNA Ladder (Invitrogen) was included in the agarose gels as a DNA weight marker.
The concentration and purity of nucleic acid solutions were estimated with NanoDrop ND‐1000 Spectrophotometer (Thermo Scientific), and their integrity was assessed by agarose gel electrophoresis.
To visualize agarose gel electrophoresed DNA molecules, gels containing GreenSafe premium (NZYTech) nucleic acid stain were imaged with ChemiDoc XRS+ Gel Imaging System (BIORAD). The 1 Kb Plus DNA Ladder (Invitrogen) was included in the agarose gels as a DNA weight marker.
10
Transformation of E. coli with Plasmid DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NEB 5-alpha competent Escherichia coli cells (New England BioLabs, Hitchin, UK) were transformed with the vector according to the manufacturer’s instructions. Briefly, 10 ng of plasmid DNA was carefully mixed with cells and incubated on ice for 30 min. Cells were heat shocked at 42 °C for 30 s and chilled on ice for 5 min. After the addition of 950 µL of SOC medium (Sigma Aldrich, St. Louis, MO, USA), the mixture was shaken at 200 rpm for 60 min at 37 °C, and the cells were diluted and plated on selective LB agar (Invitrogen, Waltham, MA, USA) plates containing 100 µg/mL ampicillin (Gibco, Waltham, MA, USA). After incubation overnight at 37 °C, selected colonies were inoculated into 3 mL of fresh LB medium containing 100 µg/mL ampicillin and incubated overnight at 37 °C with shaking at 200 rpm. The vector construct with the RdRp sequence was verified by Sanger sequencing. Plasmids for subsequent transfection of lung cells were purified using a PureLink HiPure Plasmid Miniprep Kit (Invitrogen, Waltham, MA, USA).
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!