The pRF/FMDV-IRES plasmids were kindly provided to us by Dr. Hirasawa (Memorial University of Newfoundland). Reporter genes were excised from pRF/FMDV-IRES using the restriction endonucleases EcoRV (Toyobo) and HpaI (NEB). pCAGGS/FMDV-IRES was generated by inserting a reporter gene into pCAGGS/MSC(F), which was then treated with SmaI (Takara) and rAPid Alkaline Phosphatase (Roche) using Mighty Mix (Takara). DNA fragments were purified with the Big Dye XTerminator Purification kit, followed by sequencing via capillary electrophoresis on the ABI PRISM310 genetic analyzer.
Mighty mix
Manufactured by Takara Bio
Sourced in Japan
Mighty Mix is a high-quality laboratory reagent designed for efficient DNA and RNA mixing. It provides consistent and thorough sample homogenization, ensuring optimal results in molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Mighty ta cloning kit, by Takara Bio (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Kanamycin, by Fujifilm (2 mentions)
Dna ligation kit, by Takara Bio (2 mentions)
Ampicillin, by Fujifilm (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Rapid alkaline phosphatase, by Roche (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Takara Bio (1 mentions)
Ni nitrilotriacetic acid nta agarose, by Qiagen (1 mentions)
Can get signal, by Toyobo (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Lb broth, by Merck Group (1 mentions)
Isogen 2, by Nippon Gene (1 mentions)
Pet22b, by Merck Group (1 mentions)
Spei hf, by New England Biolabs (1 mentions)
Cutsmart, by New England Biolabs (1 mentions)
Trehalose dehydrate, by Fujifilm (1 mentions)
15 protocols using mighty mix
1
Cloning and Sequencing FMDV-IRES Plasmids
2
Genetic Manipulation of E. coli Strains
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RAW264.2 cells were purchased from ATCC. All plasmids and bacterial strains used in this study are shown in Tables 2 to 4 . E. coli K-12 BW25113 (wild type [WT]) and single gene-deleted mutant strains (ΔompA: opmA::kan for JW0940-KC, ΔfepA: fepA::kan for JW5086-KC, ΔcirA: cirA::kan for JW2142-KC, ΔdegP: degP::kan for JW0157-KC, and ΔompC: ompC::kan for JW2203-KC) were provided by National BioResource Project (NIG, Japan). The competent cell BL21(DE3) strain and DH5α strain were purchased from TaKaRa (Shiga, Japan). LB broth was purchased from Sigma/Merck (Tokyo, Japan). The plasmid pET22b(+) was purchased from Novagen/Merck (Tokyo, Japan). pTV118N vector was gifted by Ishijima (Kyoto Prefectural University). Ex Taq, Mighty TA-cloning kit containing pMD T vector, DNA ligation kit Mighty Mix, in-fusion HD cloning kit, isopropyl β-d -1-thiogalactopyranoside (IPTG), and TB Green premix Ex Taq II were purchased from TaKaRa (Shiga, Japan). Ni-nitrilotriacetic acid (NTA) agarose was purchased from Qiagen (Hilden, Germany). cOmplete Mini protease inhibitor was purchased from Roche/Nippon Gene (Tokyo, Japan). Anti-OmpC antibody was purchased from MyBioSource (Vancouver, Canada). Isogen II was purchased from Nippon Gene (Tokyo, Japan). Can Get Signal and ReverTra Ace quantitative PCR (qPCR) reverse transcription (RT) kit were purchased from Toyobo (Osaka, Japan).
3
Oligomer Sequence and Reagents for DNA Cloning
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Nucleotide sequences of DNA oligomers prepared for this study were as follows, QGA adaptor antisense strand: 5’-Phosphate-CTAGAAGCGGCCGCGAATTC-(dA)18-3’, QGA adaptor sense strand: 5’-TTGAATTCGCGGCCGCTT-3’, 100-bp upstream primer: 5’-AACCTATAAAAATAGGCGTATCAC-3’, and 200-bp downstream primer: 5’-CCCCTGATTCTGTGGATAACCGTATTACCG-3’. Restriction endonucleases, XbaI (20 U/μl), SpeI-HF (20 U/μl), SpeI (10 U/μl), EcoRI (20 U/μl), and solution for digestion (CutSmart) were purchased from New England Biolabs Japan Inc (Tokyo, Japan). DNA polymerase, KOD-Plus-Neo, and the solution for PCR were purchased from TOYOBO Co Ltd (Osaka, Japan). The ligation kit, Mighty Mix, was purchased from TaKaRa BIO INC (Shiga, Japan). Triton X-100 was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Trehalose dehydrate was purchased from Wako Pure chemical Industries Ltd (Osaka, Japan). The magnetic beads, SiMAG-Oligo-dT, were purchased from Chemicell (Berlin, Germany). The neodymium magnet, ND-8R, was purchased from Magna Co Ltd (Tokyo, Japan). The kit for purification of DNA fragments, FastGene Gel/PCR Extraction Kit, was purchased from NIPPON Genetics Co Ltd (Tokyo, Japan).
4
Inverse PCR for Bovine Genome Sequencing
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The procedure for iPCR was performed as previously reported (Murakami et al., 2011 (link)). Tumor tissue DNA samples that were used for genomic PCR were digested with BclI, PstI, or BssHII and were then self-ligated using Mighty Mix (Takara Bio, Shiga, Japan). The resulting products were used as templates for PCR using inverse primers. PCR products were electrophoresed in an agarose gel, and positive samples were cloned into a pCR2.1-TOPO vector (Thermo Fisher Scientific, Waltham, MA). The product was then sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit with an Applied Biosystems 3130 Genetic Analyzer (Thermo Fisher Scientific). The bovine genome sequence adjacent to the 5′ long terminal repeat was determined using the University of California, Santa Cruz Cow BLAT Search (https://genome.ucsc.edu/cgi-bin/hgBlat?command=start) against the October 2011 freeze of the cow genome sequence as previously described (Miyasaka et al., 2015 (link)).
5
Overexpression of CrLAT1 in Chlamydomonas
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To create cDNA clones for CrLAT1 OE, RNA was prepared by the phenol/chloroform method from C. reinhardtii strain CC-408. Reverse transcription was conducted with Superscript II reverse transcriptase (Invitrogen, Waltham, MA, USA) to obtain cDNA. CrLAT1 was amplified by PCR from cDNA using PrimeSTAR GXL DNA Polymerase (Takara, Shiga, Japan) and the primer sets containing PstI and EcoRI restriction sites are shown in Supplemental Table S7 . The PCR products were cloned into pZErO-2 (Invitrogen). To construct the OE plasmid, PstI and EcoRI restriction sites were replaced with the truncated miRNA precursor cre-MIR1157 in pChlamiRNA3 (Molnar et al., 2009 (link)). After DNA sequencing, the PCR products and the modified pChlamiRNA3 vector were digested with restriction endonucleases PstI and EcoRI and ligated using Mighty Mix (Takara). Additional DNA sequencing was carried out to confirm the vector construct, which was then transformed into C. reinhardtii strain CC-4533 by electroporation as described (Iwai et al., 2014 (link)). The modified pChlamiRNA3 vector, which resulted in removal of the truncated miRNA precursor cre-MIR1157, was used as the VC. Transformants were selected on TAP plates with paromomycin (10 μg mL−1).
6
Generating Yeast GFA1 Mutant Library
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Example 2
Using error-prone PCR that generates random point mutations, a GFA1 mutant library of yeast (Saccharomyces cerevisiae) was produced. Specifically, yeast GFA1 was cloned into a p415TEF plasmid, which was then used as a template for error-prone PCR using a Genemorph II Random Mutagenesis kit (Agilent, CA, USA). The PCR amplification product was ligated into a p415TEF plasmid using Mighty Mix (Takara), and the plasmid was transformed into highly competent E. coli DH5α HIT competent cells (RBC, Banqiao City, Taipei County, Taiwan) to maximally recover the plasmid.
23 E. coli HAD phosphatases excluding HAD11 were cloned into p413GPD plasmids. For production of a HAD phosphatase library, the plasmids comprising the HAD phosphatase-encoding genes were separated and then mixed together.
7
Detailed Inhibition Assay Protocols
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FQs, CIP, LVX, and MOX that were used in inhibition assays were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Kanamycin and ampicillin were purchased from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan). Restriction enzymes and lambda DNA-HindIII DNA marker were obtained from New England Biolabs, Inc. (Ipswich, MA). DNA ligation kit, Mighty Mix, and Mighty TA cloning kit were purchased from TaKaRa Bio Inc. (Shiga, Japan). Relaxed pBR322 DNA was purchased from John Innes Enterprises Ltd. (Norwich, United Kingdom). Luria-Bertani (LB) broth (Lennox) and LB agar were purchased from Sigma (St. Louis, MO, USA). Agarose S was purchased from Nippon Gene (Toyoma, Japan). Agarose I was obtained from Dojindo (Kumamoto, Japan). Gel red was obtained from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan).
8
Lentiviral Vector Production for Gene Delivery
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The third generation lentiviral vector, CSII-EF1α-MCS, was kindly provided by Dr Miyoshi (RIKEN BRC). MyoD, GAA, and TFEB were cloned into the EcoRI site of the multiple cloning sites by overnight ligation using Mighty Mix (Takara Bio, Japan). As described previously,11 (link) large-scale culture of HEK293T cells was performed to collect viral supernatant. The virus was then concentrated by ultra-centrifugation using Centricon (EMD Millipore, Germany). Viral titers were determined using a p24 enzyme-linked immunosorbent assay kit (Cell Biolabs, San Diego, CA).
9
Cloning and Sequencing of Lactase Gene
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The amplified DNA fragments of F1/R1 and F2/ R2 were localized on the reference sequence, and the cloned regions could not completely cover the entire lactose gene and their sizes were approximately 500 bp (Figure 1), so the complete lactase gene could not be amplified. Furthermore, the amplified DNA fragments of F/R, F3/R3, and F4/R4 were analyzed, and they all contained the enzyme gene (Figure 1), but no amplification product was observed through agarose gel electrophoresis. Therefore, these primers (F1/R1, F2/ R2, F/R, F3/R3, and F4/R4) were recombined to ultimately yield the enzyme gene.
The templates were amplified with PCR using primer pairs (Table 2), and primer F5/R1 was used to test the homology between the lactase gene of the experimental strain and the reference sequence to determine the PCR primers. The target gene (bgaB) was amplified by PCR with PrimeSTAR HS DNA Polymerase, and amplification products were detected by 1% agarose gel electrophoresis, purified (Agarose Gel DNA Purification Kit Ver. 2.0) and analyzed (TaKaRa Biotechnology).
According to the operational instructions of the DNA Ligation Kit < Mighty Mix > (TaKaRa Biotechnology), bgaB was linked to pET-32a(+) (Figure 2). Then, pET32-bgaB was transformed into E. coli HST08 and cloned. Finally, the plasmids were extracted from the positive clones and sequenced.
The templates were amplified with PCR using primer pairs (Table 2), and primer F5/R1 was used to test the homology between the lactase gene of the experimental strain and the reference sequence to determine the PCR primers. The target gene (bgaB) was amplified by PCR with PrimeSTAR HS DNA Polymerase, and amplification products were detected by 1% agarose gel electrophoresis, purified (Agarose Gel DNA Purification Kit Ver. 2.0) and analyzed (TaKaRa Biotechnology).
According to the operational instructions of the DNA Ligation Kit < Mighty Mix > (TaKaRa Biotechnology), bgaB was linked to pET-32a(+) (Figure 2). Then, pET32-bgaB was transformed into E. coli HST08 and cloned. Finally, the plasmids were extracted from the positive clones and sequenced.
10
Cloning Antioxidant Response Element
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A sequence-encoding ARE was inserted at XhoI and HindIII sites of the pGL4.28[luc2CP/minP/Hygro] vector (Promega, Madison, WI, USA). The inserted sequences were
ARE-GST-Ya-F
(5′-TCGAGTAGCTTGGAAATGACATTGCTAATGGTGACAAAGCAACTTTA-3′; underlining marks the ARE consensus sequence) and
ARE-GST-Ya-R
(5′-AGCTTAAAGTTGCTTTGTCACCATTAGCAATGTCATTTCCAAGCTAC-3′) [49 (link)].
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