For construction of Flag-tagged or hemagglutinin (HA)-tagged mammalian expression plasmids, cDNA of PCBP2, cGAS, STING, TBK1, or IRF3 was amplified using TransStart® FastPfu DNA Polymerase (TransGen Biotech, Cat# AP221-01), and inserted into pCDH or pcDNA3.0 vector. For construction of histidine (His)-tagged cGAS or glutathione S-transferase (GST)-tagged PCBP2 bacterial expression plasmids, cDNAs were subcloned into pET-28a or pGEX4T-1 vector, respectively. To construct mammalian expression plasmids encoding GFP-tagged cGAS, mCherry-tagged PCBP2, spGFP1-10-V5-cGAS, PCBP2-2×spGFP11, cGAS-2×spGFP11, DNA fragments encoding the tag protein, PCBP2 or cGAS were amplified and assembled to linear pCDH vector using ClonExpress MultiS One Step Cloning Kit (Vazyme, Cat# C113-02); and DNA fragments encoding GFP-cGAS or mCherry-PCBP2 were assembled to linear pET-28a vector to generate the bacterial expression plasmids. PCBP2 and cGAS mutants were generated by PCR-based mutagenesis using 2x Phanta Max Master Mix (Vazyme, Cat# P525-03). IFNβ-, ISRE-luciferase (Luc) reporter plasmids were kindly provided by Dr. Hongbing Shu41 (link), and NF-κB-Luc reporter plasmid was generously provided by Dr. Zhijian Chen42 (link).
Phanta max master mix
Manufactured by Vazyme
Sourced in China
Phanta Max Master Mix is a high-performance PCR master mix developed by Vazyme. It is designed for fast, reliable, and efficient DNA amplification in various PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Clonexpress 2 one step cloning kit, by Vazyme (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Miseq platform, by Illumina (4 mentions)
Rapid taq master mix, by Vazyme (4 mentions)
Pmd18 t vector, by Takara Bio (4 mentions)
Clonexpress ultra one step cloning kit, by Vazyme (4 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (4 mentions)
Quant it picogreen kit, by Thermo Fisher Scientific (3 mentions)
Expicho expression system, by Thermo Fisher Scientific (3 mentions)
Fastpure cell tissue dna isolation mini kit, by Vazyme (3 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (3 mentions)
Miseq reagent kit, by Illumina (2 mentions)
Taq master mix, by Vazyme (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Tcs sp8, by Leica (2 mentions)
Peasy blunt cloning vector, by Transgene (2 mentions)
Hiscript 2 one step rt pcr kit, by Vazyme (2 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (2 mentions)
Fastpure plasmid mini kit, by Vazyme (2 mentions)
Eastep super total rna extraction kit, by Promega (2 mentions)
114 protocols using phanta max master mix
1
Molecular Cloning and Expression Constructs
2
Detecting SHARPIN Gene Hotspot Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from the blood using the phenol-chloroform method (24 (link)). The FFPE genomic DNA was extracted using a QIAamp DNA FFPE Tissue kit (Qiagen GmbH, Hilden, Germany). To detect hotspot mutations, 8 exons and exon-intron adjacent sequences of the SHARPIN gene were amplified using PCR. In the DNA from the tumor samples, each amplification reaction was performed under standard conditions in a 20 µl PCR mixture containing 70–150 ng template DNA, 10 pmol primers, and 10 µl 2X Taq Master Mix (Dye Plus) (Vazyme, Piscataway, NJ, USA). The GC percentage of Exon 1 was relatively high; therefore, the 2X Taq Master Mix (Dye Plus) was replaced by 2X Phanta Max Master Mix (Vazyme) in the amplification of Exon 1. The 8 primer pairs that were used are listed in Table I . Exon 3 was amplified by PCR. The thermocycler conditions for the standard and nested PCR protocols are listed in Table II . PCR products were purified using QIAquick reagent (Qiagen GmbH) and directly sequenced based on the Big Dye Terminator sequencing chemistry (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA USA) in an ABI3130 automated sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.). All mutations were confirmed through repeated bidirectional sequencing on the ABI sequencer. Gene sequences were blasted using DNASTAR Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA).
3
16S rDNA Illumina Sequencing Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The V3-V4 region, a 468 bp within the 16 s rDNA gene, was used to build the illumine sequencing library and amplified with the broadly conserved primers 341F (5′–CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). Different identifier codes were added at each primer for the further illumina sequencing. Polymerase chain reaction (PCR) was applied in a 50 μl reaction system including 25 μl 2x Phanta Max Master Mix (Vazyme, China), 10 mM each primer, 16 μl each ddH2O and 5 μl DNA template. The PCR program was initial denaturation at 95 °C, with 8 cycles of denaturation at 95 °C for 30s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, with a final elongation phase at 72 °C for 5 min. The PCR products were analyzed by Quant-It Pico Green kit (Invitrogen, United States) and library was prepared. Barcoded samples were combined equal concentrations according to volume of sequencing. The library concentration was measured using Agilent 2100 Bioanalyzer (Agilent Technologies, United States), and followed by elution with Tris_HCl (pH 8.5). After denaturation, barcoded samples were combined following the volume of sequencing and sequenced on a PE250 v3 instrument using 600 cycles MiSeq Reagent Kit on a MiSeq Platform (Illumina; United States).
4
Plasmid-safe DNA Digestion and PCR
5
RNA-seq Library Preparation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extracted RNA was treated with DNase to eliminate any residual genomic DNA, after which magnetic beads with oligo-(dT) were used to enrich the eukaryotic mRNA. The mRNA was broken into short fragments. The disrupted mRNA and random hexamer primers were used to synthesize first-strand cDNA. After synthesizing the second cDNA strand, the double-stranded cDNA was purified using a commercial kit (Promega, Madison, WI, United States). The purified cDNA underwent an end-repair step before a poly-A tail and a sequencing adapter were added. Following a size selection step, the sequences were amplified by PCR with 2 x Phanta Max Master Mix (Vazyme, Nanjing, China). The libraries were qualitatively analyzed using the Agilent 2,100 Bioanalyzer (Agilent, Santa Clara, CA, United States) before they were sequenced using the Illumina HiSeq™ 2,500 system (Illumina, San Diego, CA, United States), which generated 125 or 150 bp paired-end reads.
6
Identifying SNP in Equine TOE1 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PCR-ACRS method was designed to identify the single nucleotide polymorphism that determined the CA occurrence (rs397160943, NC_009145.3:g.13122415C>T; ENSECAT 00000024892.2:c.284G>A, ENSECAP00000020698.1:p.Arg95His). The primers were designed using Primer3 Input (version 0.4.0) and TOE1 gene (ENSECAG00000023204) reference (Table 1 ), and due to the modified primer sequence, the artificial restriction site for HpyCH4III (BioLabs, New England, Ipswich, MA, USA) was created. The PCR was obtained using 2xPhanta Max Master Mix (Vazyme, Polgen, Łódź, Poland), and after 16 h digestion, the products were obtained as follows: C allele-111, 22 bp and T allele-133bp.
7
Genotyping Mouse Tail DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from mouse tail tissue using the Trelief Animal Genomic DNA Kit (TsingKe Biotech, Beijing, China). Genotypes were determined by PCR using 2× Phanta Max Master Mix (Vazyme, Nanjing, China) using the following primers: 5′-gcatcgcagccagtggtgtt-3′ (forward) and 5′-gggcattaggaggcaaatgaaatt-3′ (reverse). DNA electrophoresis was done on 1% agarose gels with wildtypes and homozygotes producing single bands of 1306 and 599 bp, respectively. Heterozygotes produced both bands.
8
Recombinant DNA Manipulation in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The strains used in this study were listed in Supplementary Table 1 , E. coli DH5α served as the host for recombinant DNA manipulation and plasmid construction. For cis-3-HyPip production, E. coli BL21(DE3) and derived strains were used. The plasmid pEcgRNA carrying the ccdB gene was constructed and maintained in E. coli DB3.1. The DNA polymerases used for polymerase chain reaction (PCR) including 2 × Phanta Max Master Mix, 2 × Taq Master Mix and ClonExpress One Step Cloning Kit were purchased from Vazyme (Nanjing, China). Molecular biological reagents, such as T4 DNA ligase and DNA gel extraction kit, were obtained from TaKaRa (Dalian, China). Isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, spectinomycin, and kanamycin were provided from Sangon Biotech (Shanghai, China). All other chemicals were purchased from Sigma-Aldrich (Shanghai, China) or Sangon Biotech and were of analytical grade (Shanghai, China).
9
Cloning and Sequencing of CPR cDNA from N. lugens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CPR cDNA sequence was obtained by searching the N. lugens transcriptome database in the Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra , accessed date: 7 January 2019) (SRA accession number SRX023419) [28 (link)]. Total RNAs were extracted from the whole bodies of the various developmental stages using RNAiso Plus Total RNA extraction reagent (Takara, Dalian, China). First-strand cDNA was synthesized using HiScript® II Q RT SuperMix for qPCR with gDNA wiper Mix (Vazyme Biotech, Nanjing, China). A pair of primers (sense: 5′-ATGGAGGTGGAGGCTGACTTAG-3′; antisense: 5′-TCAACTCCATACGTCGGCCG-3′) were designed to amplify the complete open reading frame (ORF) of the CPR gene. PCR was performed using 2 × Phanta® Max Master Mix (Vazyme Biotech, Nanjing, China). The PCR product was cloned into the pMD19-T vector (Takara, Dalian, China) and sequenced by Sanger sequencing (Tsingke Biotech, Beijing, China).
10
Identification and Verification of HMGR Genes in Lithospermum erythrorhizon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Pfam domain PF00368 is found in Pfam protein family databases (http://pfam.xfam.org/ , accessed on 25 April 2023) and HMMER 3.0 (https://github.com/PhyreEngine/conda-hmmer , accessed on 25 April 2023) was used to search the genomes of 36 plants for HMGR genes. The download addresses for genome files of all species are listed in Table S1 . Then redundant sequences and abnormal sequences (incomplete PF00368 domain) identified by Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi , accessed on 25 April 2023) were removed. Eleven gene sequences of the HMGR family in L. erythrorhizon genome were obtained, then used to blast against the eight LerHMGRs from the genome published by Auber et al. using all-to-all blastp, and primers (Table S9 ) were designed for amplification with cDNA mixtures of seedling leaves and roots as template to verify all sequences in L. erythrorhizon using Phanta Max Master Mix (Vazyme, #P515, Nanjing, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!