The C-terminal FLAG-tagged mouse mGluR2 construct in pcDNA3.1(+) expression vector was purchased from GenScript (ORF clone: OMu19627D) and verified by sequencing (ACGT Inc). Full length mGluR2 construct with an amber codon (TAG) mutation of amino acid A548 (azi-CRD) or N-terminal SNAP-tag (SNAP-mGluR2) were generated as previously reported (Liauw et al., 2021 (link)). The insertion of an amber codon (TAG) between E715 and V716 in mGluR2 (azi-ECL2) was performed using the QuikChange site-directed mutagenesis kit (Agilent). SNAP-mGluR2 constructs used for calcium imaging had C-terminal FLAG-tag removed by PCR-based deletion using phosphorylated primers. All plasmids were sequence verified (ACGT Inc). DNA restriction enzymes, DNA polymerase and DNA ligase were from New England Biolabs. Plasmid preparation kits were purchased from Macherey-Nagel.
Plasmid preparation kits
Manufactured by Macherey-Nagel
Plasmid preparation kits are laboratory equipment used for the isolation and purification of plasmid DNA from bacterial cultures. These kits provide a standardized and efficient method for extracting plasmid DNA, which is a crucial step in various molecular biology applications, such as genetic engineering and DNA cloning.
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Lab products found in correlation
Dna ligase, by New England Biolabs (4 mentions)
Dna polymerase, by New England Biolabs (4 mentions)
Pcdna3.1 expression vector, by GenScript (4 mentions)
Orf clone omu19627d, by GenScript (3 mentions)
Hifi dna assembly master mix, by New England Biolabs (3 mentions)
Psnapf, by New England Biolabs (3 mentions)
Quikchange site directed mutagenesis kit, by Qiagen (3 mentions)
Omu17682d, by GenScript (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Hek293, by Leibniz Institute DSMZ (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Mycoalert kit, by Lonza (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Complete edta free, by Roche (1 mentions)
Dna restriction enzymes, by New England Biolabs (1 mentions)
5 protocols using plasmid preparation kits
1
Generation of Engineered mGluR2 Constructs
2
Routine Cell Culture and Molecular Biology Techniques
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Cell lines HEK293 (Cat# ACC-305, RRID:CVCL_0045 ) and 293T (Cat# ACC-635, RRID:CVCL_0063 ) were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Regular testing for mycoplasma contamination is performed using the MycoAlert kit from Lonza. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin and penicillin–streptomycin were purchased from Gibco (Life Technologies). DNA restriction enzymes, Phusion DNA polymerase, T4 DNA ligase and PNGase F were from New England Biolabs. Plasmid preparation kits were from Macherey-Nagel. Azi was purchased from Bachem. Protease inhibitor cOmplete EDTA-free from Roche and was used supplemented with 1 mM EDTA. Polyethylenimine MAX was from Polyscience, dissolved in H2O as 10 mg/mL stock solution, aliquoted and stored at −20°C. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and resin were purchased from Novabiochem or Iris Biotech. Chloroacetic acid (ClAcOH) was from Sigma.
3
Mutagenesis and tagging of mGluR2/3
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The C-terminal FLAG-tagged mouse mGluR2 and mGluR3 constructs in pcDNA3.1(+) expression vector were purchased from GenScript (ORF clone: OMu19627D and OMu17682D) and verified by sequencing (ACGT Inc). The mutation of amino acid A548 in mGluR2 and A557 in mGluR3 to an amber codon (TAG) was performed using the QuikChange site-directed mutagenesis kit (Qiagen). The same approach was used to introduce point mutations in mGluR2 (Y216A, D295A, C770A, and L521C) for other experiments. For the YADA/WT mGluR2 (548UAA) heterodimer experiment, a SNAP-tag (pSNAPf, NEB) was inserted at position 19, flanked by GGS linkers, using HiFi DNA Assembly Master Mix (NEB). Finally, site directed mutagenesis was done to remove the C-terminal FLAG-tag on this construct. All plasmids were sequence verified (ACGT Inc). DNA restriction enzymes, DNA polymerase and DNA ligase were from New England Biolabs. Plasmid preparation kits were purchased from Macherey-Nagel.
4
Mutagenesis and tagging of mGluR2/3
5
Engineered CaSR Mutants with SNAP-tag and mEGFP
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A mouse CaSR construct with a C-terminal FLAG-tag in pcDNA3.1+ expression vector was purchased from GenScript (ORF clone: OMu14241D) and validated by sequencing (ACGT). A SNAP-tag (pSNAPf, NEB) flanked by GGS linkers was inserted at position 21 using HiFi DNA Assembly Master Mix (NEB). Point mutations in CaSR (P55L, C129S, C131G, C129G + C131G, T151M, L174R, R220W, R227L, E228K, G557E) were introduced using a QuikChange site-directed mutagenesis kit (Qiagen). The SNAP-tag CaSR construct was used as the template for mutation of amino acid E593 to an amber codon (TAG) via QuikChange mutagenesis. AscI restriction sites were used to insert mEGFP (mEGFP-N1, gift from Michael Davidson (Addgene plasmid # 54767) at the C-terminus of the GenScript construct, and it was used as the template for mutation of amino acid D451 to an amber codon (TAG) via QuikChange mutagenesis. All plasmids were verified by sequencing (ACGT). DNA restriction enzymes, DNA polymerase, and DNA ligase were purchased from New England Biolabs. Plasmid preparation kits were obtained from Macherey-Nagel.
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