HEK293T cells were transfected twice with control siRNA or siRNAs targeting SIVA1. 24 h after the second transfection, cells were introduced with the SupF tRNA gene containing shuttle plasmid pZ189 irradiated with 300 J/m2 UV. 48 h later, the plasmid was extracted from cells using a purification kit (AxyPrep Plasmid Miniprep; Axygen). To exclude the methylated template, the purified plasmid was digested with DpnI, and then, E. coli strain MBM7070 was transformed with the processed plasmid and selected for 100 µg/ml ampicillin resistance on Luria–Bertani plates containing 100 µg/ml X-gal and 1 mM IPTG. In Fig. 4 G , white (mutant) and blue (wild type) colonies were counted, and the mutation frequency was calculated as the ratio of white colonies to total colonies. The pZ189 plasmid and MBM7070 strain were gifts from J. Shao (Zhejiang University, Hangzhou, China).
Axyprep plasmid miniprep
Manufactured by Corning
Sourced in United States
The AxyPrep Plasmid Miniprep is a laboratory equipment product designed for the rapid and efficient extraction of plasmid DNA from bacterial cultures. It is a simple and reliable tool for purifying high-quality plasmid DNA suitable for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Axyprep dna gel extraction kit, by Corning (2 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Protein ladder, by Takara Bio (1 mentions)
Primestar dna polymerase, by Takara Bio (1 mentions)
Ptrchis2b, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Genetailor site directed mutagenesis system kit, by Thermo Fisher Scientific (1 mentions)
Ppiczαa, by Thermo Fisher Scientific (1 mentions)
One step cloning kit, by Vazyme (1 mentions)
4 protocols using axyprep plasmid miniprep
1
UV-Induced Mutation Assay in HEK293T Cells
2
Cloning and Characterization of Trc Promoter Library
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All primers and plasmids used in this study are listed in Table1 . E. coli DH10B (Promega, Beijing, China) was used for plasmid construction, promoter library construction, and characterization. E. coli BL21(DE3) was used for peptide expression. Plasmid pTrcHis2B (Invitrogen, Shanghai, People's Republic of China) harboring trc promoter and its mutated promoter library was constructed and preserved in our laboratory. PrimeStar DNA polymerase, restriction endonucleases, T4 DNA ligase, and protein ladder were purchased from Takara Biotechnology (Dalian, People's Republic of China). Plasmid DNA isolation and DNA gel purification were performed using AxyPrep Plasmid Miniprep and AxyPrep DNA gel extraction kits (Axygen Biosciences, Union City, USA). DNA primers, bovine serum albumin (BSA), and all other regents were provided by Sangon Biotech (Shanghai, People's Republic of China). The working concentration of ampicillin was 100 mg L−1.
3
Site-Directed Mutagenesis of LCAT
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We used the GeneTailor Site-Directed Mutagenesis System kit (Invitrogen, California, USA) to obtain plasmids containing either the c.997G > A (p.V333 M) or c.1210A > G (p.M404 V) cDNA sequences, to be used as expression vectors for in-vitro cellular assays. Mutagenesis was performed using the pCMV6-XL4 circular vector carrying the cDNA sequence of LCAT (OriGene, Maryland, USA). Primers for the c.997G > A variant were: forward 5′-GCAGGACTCCCAGCACCTGGTATGGAAGTATAC-3′ and reverse 5′-ACCAGGTGCTGGGAGTCCTGCCAGGAGGTCACG-3′. Primers for the c.1210A > G variant were: forward 5′-CGGGATACAGCATCTCAACGTGGTCTTCAG-3′ and reverse 5′-GTTGAGATGCTGTATCCCGTGCAGGGGCAGC-3′. After transformation in E. coli DH5αTM-T1® (Thermo Fisher Scientific, California, USA), plasmid DNA was purified using AxyPrep Plasmid Miniprep (Axygen Biosciences, California, USA). Plasmid integrity was verified by enzymatic digestion with FastDigest® SmaI restriction enzyme (Fermentas, California, USA) and visualized on a 1% agarose gel. The presence of both c.997G > A and c.1210A > G variants were confirmed by direct sequencing using specific primers for this vector (VP1.5 and XL39, OriGene, Maryland, USA).
4
Engineered Microbial Phytase Production
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The gene encoding parental E. coli AppA phytase was synthesized by Sangon Biotech (Shanghai, China). Random mutagenesis kits for epPCR were obtained from Agilent (California, United States). The AxyPrep plasmid miniprep and AxyPrep DNA gel extraction kits were purchased from Axygen (California, United States). The One Step Cloning Kit was obtained from Vazyme (Nanjing, China). All other chemicals and reagents were purchased from Sangon (Shanghai, China) and were of analytical grade. The expression vector pET28a in E. coli was obtained from Novagen (Madison, Wisconsin, United States). The expression vector pPICZαA (Invitrogen, United States ) in P. pastoris was obtained from Invitrogen (San Diego, United States). E. coli BL21 (DE3) was used as the host for screening, while P. pastoris was used for recombinant protein expression and characterization.
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