Rosetta 2 cells containing pET22b-U2 were obtained in house.[8 (link)] Degenerate primers for site saturation mutagenesis of position 49 were purchased from Integrated DNA Technologies (Skokie, Illinois, USA). Dpn1 enzyme was purchased from New England Biolabs (Ipswich, Massachusetts, USA). Saccharomyces cerevisiae tRNAPhe, terrific broth media, hexafluoroisopropanol (HFIP), and ammonium acetate solution were obtained from Sigma Aldrich (St. Louis, Missouri, USA). Carbenicillin, chloramphenicol, Isopropyl-β-D-thiogalactoside (IPTG), and lysozyme were purchased from Gold Biotechnology (St. Louis, Missouri, USA). The Ni-NTA His-Bind purification kit and 4–20% SDS gels were purchased from Novagen (Madison, Wisconsin, USA). Hydrochloric acid, 15% TBE-urea gels, Tris-Cl, boric acid, and triethylamine (TEA) were acquired from Fisher Scientific (Waltham, Massachusetts, USA). The synthetic 12-mer oligonucleotide was purchased from Dharmacon (Lafayette, Colorado, USA).
Dpn1 enzyme
Manufactured by New England Biolabs
Sourced in United States
Dpn1 enzyme is a restriction endonuclease that recognizes and cleaves the DNA sequence 5'-Gm6ATC-3'. It is commonly used in molecular biology for the identification and removal of methylated DNA, which is often generated during bacterial plasmid replication.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium acetate solution, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Carbenicillin, by GoldBio (1 mentions)
Boric acid, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by GoldBio (1 mentions)
Lysozyme, by GoldBio (1 mentions)
Saccharomyces cerevisiae trnaphe, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by GoldBio (1 mentions)
Tris cl, by Thermo Fisher Scientific (1 mentions)
Hexafluoroisopropanol hfip, by Merck Group (1 mentions)
Triethylamine tea, by Thermo Fisher Scientific (1 mentions)
Quikchange lightening kit, by Agilent Technologies (1 mentions)
Hifi dna assembly master mix, by New England Biolabs (1 mentions)
Neb stable competent e coli, by New England Biolabs (1 mentions)
Phusion flash polymerase, by Thermo Fisher Scientific (1 mentions)
Dna clean and concentrator 5 column, by Zymo Research (1 mentions)
Tianprep mini plasmid kit, by Tiangen Biotech (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
4 protocols using dpn1 enzyme
1
Site-Directed Mutagenesis Protocol for Protein Engineering
2
Directed Mutagenesis of Hrp1 Protein
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Hrp1 protein mutants (K160E, D193N, L205S, W168F, W168A, F162W, and F204W) were created using the QuikChange Lightening Kit based on manufacturer guidelines for primer design and PCR (Agilent). The PCR template was the pRS313-HRP1 plasmid, which includes −500 to +1,848 of sequence relative to the HRP1 + 1 ATG. The PCR product was digested with Dpn1 enzyme prior to transformation into E. coli 5-alpha competent cells (New England Biolabs). The E. coli transformants were screened by plasmid purification and sequencing to confirm the presence of desired mutations.
3
CRISPR Targeting Site Identification and Assembly
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Geneious Prime software (Biomatters, Auckland, New Zealand) was used to identify high off-target score CRISPR targeting sites near the SOX2 and SIX6 stop sites as well as the CLYBL safe harbor site (Table S2 ). PCR amplifications were carried out with Phusion Flash Polymerase (#F548L; Thermo Fisher Scientific, Waltham, MA, USA) and appropriate oligos (Tables S1 and S4 ). PCR products were column purified using DNA Clean and Concentrator-5 columns (#D4014; Zymo Research, Irvine, CA, USA). DNA fragments were stitched together using HiFi DNA Assembly Mastermix (#E2621S; NEB, Ipswich, MA, USA), and parental plasmids were removed with the methylation-sensitive Dpn1 enzyme (#R0176L; NEB, Ipswich, MA, USA). For all work except for minicircle production, plasmids were transformed into NEB Stable Competent E. coli (#C3040I; NEB, Ipswich, MA, USA) cells.
4
Site-directed Mutagenesis of AAV Plasmid
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Serine (S)→alanine (A) and lysine (K)→arginine (R) mutations were introduced into AAV-ie rep/cap plasmid with Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Cat.P505) according to the manufacturer’s protocol. Briefly, a one-step PCR amplification of the target sites was performed for 28 cycles with the primers (Supplementary Table S1 ). The PCR products were digested by Dpn1 enzyme (NEW ENGLAND Bio Labs, cat. R0176S) for 2 h. Then ten microliters of the recombinant product were transformed into DH5α chemically competent cell (Shanghai Weidi Biotechnology CO., Ltd, cat. DL1001). Plasmids were isolated with TIANprep Mini Plasmid Kit (TIANGEN BIOTECH(BEIJING) CO., LTD, cat.DP118) and verified by DNA sequencing (Applied Biosystems 3730XL genetic analyzer; BioSune, Shanghai, China).
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