Purelink expi endotoxin free maxi plasmid purification kit

Manufactured by Thermo Fisher Scientific

The PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit is a laboratory tool designed for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to efficiently capture and elute plasmid DNA, while removing endotoxins and other contaminants.

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Lab products found in correlation

Hek293t, by American Type Culture Collection (1 mentions) Calphos mammalian transfection kit, by Takara Bio (1 mentions) Nu j mice, by Jackson ImmunoResearch (1 mentions) Syringe pump, by KD Scientific (1 mentions) Stereotaxic frame, by RWD Life Science (1 mentions) Femtojet 4, by Eppendorf (1 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Neb q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) 10 beta competent e coli cells, by New England Biolabs (1 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)

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PureLink kit (67 citations) Plasmid purification kits (7 citations) PureLink™ Plasmid Purification Kit (3 citations)

8 protocols using purelink expi endotoxin free maxi plasmid purification kit

Lentivirus and AAV Packaging Protocols

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For lentivirus packaging, pLL3.7m-based
plasmids were purified by PureLink Expi Endotoxin-free Maxi plasmid
Purification kit (Invitrogen), and then HEK293T (ATCC, No. CRL-3216)
cells at ∼70% confluency were transfected with psPAX2, pMD2.G,
and pLL3.7m plasmids using CalPhos mammalian transfection kit (Takara
Bio). Two days following transfection, viral supernatant was filtered
with a 0.45 μm PES filter before using to infect target cells.
For AAV packaging, pAAV-CMV-tKiMBI-T2A-caMEK-bGH_pA plasmids were
purified by PureLink Expi Endotoxin-free Maxi plasmid Purification
kit (Invitrogen) and then were sent to Stanford Gene Vector and Virus
Core to produce and titer AAV.DJ vectors.

Wu Y., Walker J.R., Westberg M., Ning L., Monje M., Kirkland T.A., Lin M.Z, & Su Y. (2023). Kinase-Modulated Bioluminescent Indicators Enable Noninvasive Imaging of Drug Activity in the Brain. ACS Central Science, 9(4), 719-732.

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Pcpe2 Expression Vector Construction

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Mouse Pcpe2 was cloned into a mammalian expression vector under the control of the CMV promoter. The Gaussia luciferase secretion signal was used at the N-terminus of the construct and a GGGGS linker was added at the C-terminus immediately followed by an 8xHis tag (TOP Gene Technologies Inc). Expansion of the plasmid was carried out using PureLink ExpiEndotoxin-Free maxi plasmid purification kit (Invitrogen).

Xu H., Thomas M.J., Kaul S., Kallinger R., Ouweneel A.B., Maruko E., Oussaada S.M., Jongejan A., Cense H.A., Nieuwdorp M., Serlie M.J., Goldberg I.J., Civelek M., Parks B.W., Lusis A.J., Knaack D., Schill R.L., May S.C., Reho J.J., Grobe J.L., Gantner B., Sahoo D, & Sorci-Thomas M.G. (2021). Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated HDL Uptake. Arteriosclerosis, thrombosis, and vascular biology, 41(11), 2708-2725.

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In Vivo Bioluminescence Imaging of AAV

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For AAV packaging, pAAV-CMV-AkaLuc-P2A-mNeonGreen-WPRE plasmid and pAAV-CMV-Antares-P2A-mNeonGreen-WPRE were amplified and purified by a PureLink Expi endotoxin-free maxi plasmid purification kit (Invitrogen) and were sent to Stanford Gene Vector and Virus Core to produce and titer AAV.DJ vectors. Under sterile conditions, 12-week-old male J:NU mice (Jackson Laboratory, 007850) were anesthetized with isoflurane and secured in a stereotaxic frame (RWD Life Science), and a hole the size of the needle was drilled through the skull. A Hamilton syringe with a 33-gauge needle was inserted at anteroposterior −2.2 mm, mediolateral +1.7 mm and dorsoventral −1.7 mm. After a 2-min wait, the needle was pulled back 0.3 mm, and 1 µl containing 1.4 × 10¹⁰ genome copies each of AkaLuc- and Antares-expressing AAV was injected at a speed of 0.1 μl min^–1 using a syringe pump (KD Scientific). The needle was left in place for 3 min after each injection to minimize upward flow of viral solution. Two to 3 weeks after AAV infection, mice were injected i.p. with the indicated amount of CFz, Akalumine or FFz and imaged on the Ami HT (binning of 1 × 1 and exposure time of 10 s).

Su Y., Walker J.R., Hall M.P., Klein M.A., Wu X., Encell L.P., Casey K.M., Liu L.X., Hong G., Lin M.Z, & Kirkland T.A. (2023). An optimized bioluminescent substrate for non-invasive imaging in the brain. Nature Chemical Biology, 19(6), 731-739.

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Efficient Plasmid Microinjection for Culex Mosquitoes

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The injected DNA plasmids were prepared using PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Invitrogen, Cat.# A31231), aliquoted based on the concentrations outlined in the manuscripts, and later stored at −80 °C before proceeding to microinjection. All injections were performed on a microinjection station equipped with a FemtoJet 4 microinjector (Eppendorf). The prepared Cas9/sgRNA mixtures were injected into the posterior end of Culex quinquefasciatus embryos eggs freshly collected after oviposition (~1 h) to ensure efficient targeting of the germline.

Feng X., López Del Amo V., Mameli E., Lee M., Bishop A.L., Perrimon N, & Gantz V.M. (2021). Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes. Nature Communications, 12, 2960.

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Culex quinquefasciatus Embryo Microinjection

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The Culex quinquefasciatus embryo collection and microinjection followed our previously established procedures 30 (link) . The injected DNA plasmids were prepared using PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Invitrogen, # A31231), aliquoted based on concentrations, and later stored at -80℃ for microinjection.

Feng X., Amo V.L.D., Mameli E., Lee M., Bishop A.L., Perrimon N., & Gantz V.M. (2021). Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development.

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Plasmid Cloning and Sanger Sequencing

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forward, 5’- CTCACGCCTGCCCTCTTGCCTGC-3’
reverse, 5’-TGGGGAGGCCGTGGCTGG-3’
Following transformation, clones were selected, and the plasmid DNA was Sanger sequenced to confirm clones with the correct sequences. Vectors with the correct sequence were purified using the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Thermofisher Scientific) and the sequence was confirmed again using Sanger sequencing. The vector DNA with either the risk or non-risk allele was stored at −20°C until the iPSCs were ready to be transfected.

Sonti S., Littleton S.H., Pahl M.C., Zimmerman A.J., Chesi A., Palermo J., Lasconi C., Brown E.B., Pippin J.A., Wells A.D., Doldur-Balli F., Pack A.I., Gehrman P.R., Keene A.C, & Grant S.F. (2023). Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q. bioRxiv.

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Cloning and Mutagenesis of GC-Rich Enhancer

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forward, 5’-TTTCTCTATCGATAGGTTCGGCCCCTCCCCAGGCCCCTCCCCGCCCCCCCCCCCCCCCGG-3’
reverse, 5’- TCGAGCCCGGGCTAGGGCCTCCACCCTCCCTCC-3’.
The amplification of the putative enhancer containing rs9932282 proved challenging given the high GC rich content. We changed the primer design to incorporate as much of the GC rich region as we could into one oligo, resulting in a 60 bp oligo.
Following infusion cloning, the recombinant DNA was transformed into NEB Stable E. coli cells (NEB) under standard transformation conditions. Colonies were selected and plasmid DNA was extracted using The PureLink^™ Quick Plasmid Miniprep Kit (Thermofisher Scientific) and sent for Sanger sequencing to identify the integrity and quality of the inserted enhancer fragment. Vectors with the correct sequence were purified using the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Thermofisher Scientific) and the sequence was confirmed again using Sanger sequencing. Once the putative enhancers containing all three SNPs were successfully cloned into the pGL3 vector, site directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) and the NEB Q5 Site Directed Mutagenesis kit (NEB) was performed to introduce the risk allele for each SNP. The primers used for mutagenesis are as follows:

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Transfection of Manf siRNA Lentivectors

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Scrambled siRNA-GFP and Manf siRNA-GFP lentivector plasmids were purchased from Applied Biological Materials (Richmond, BC, Canada) with the MANF siRNA target sequence 5′-TCAAAGACAGAGATGTCACATTTTCACCA-3′ cloned into the piLenti-siRNA-GFP plasmid backbone. The siRNA is driven by the U6 promotor followed by the GFP driven by the CMV promotor. The plasmids were subcloned and amplified in 10-beta competent E. coli cells (New England Biolabs), and purified using PureLink Expi Endotoxin-free Maxi Plasmid Purification Kit (Thermo Fisher Scientific). Either 2.5 μg of scrambled siRNA or Manf siRNA lentivector plasmids were transfected into N2a or SH-SY5Y cells using Lipofectamine 3000 Reagent (Life Technologies) according to the manufacturer’s instruction. Cells were subject to subsequent analysis.

Wen W., Wang Y., Li H., Xu H., Xu M., Frank J.A., Ma M, & Luo J. (2020). Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways. Frontiers in Molecular Neuroscience, 13, 560020.

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