To generate a translational reporter, a 2.7-kb fully encoding genomic fragment of MSL10 (gMSL10) was cloned via PCR (PrimerStar Max; Takara) from previously extracted genomic DNA from Col-0. A 720-bp fragment encoding mEGFP was subsequently cloned in-frame with a 135-bp terminator from sequence of MSL10 via PCR. The binary vector pBIN30 was linearized via “inside-out” PCR (PrimerStar Max; Takara) for later blunt end ligation with the cloned fragments. pMSL10 and gMSL10 fragments were fused to the mEGFP-terminator sequence via a flexible GLY-GLY-SER-GLY linker. The construct was ligated into the pBIN30 binary vector with In-Fusion ligation technology (Takara). The full sequence of the complementation/reporter construct has been submitted to National Center for Biotechnology Information (NCBI) GenBank (accession code MZ380291). Successful Agrobacterium-mediated transgenesis of msl10-1 plants harboring the complementation construct was evaluated by BASTA resistance and GFP fluorescence in roots.
Primer star max
Manufactured by Takara Bio
Sourced in Japan
Primer STAR Max is a high-fidelity DNA polymerase designed for accurate and efficient DNA amplification. It features a proofreading function to minimize errors during PCR, enabling the generation of reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Protein marker, by Thermo Fisher Scientific (2 mentions)
Plasmid purification kit, by Takara Bio (2 mentions)
Dna marker, by Takara Bio (2 mentions)
Dna gel extraction kit, by Takara Bio (2 mentions)
M9 minimal salts m9 buffer, by Sangon (2 mentions)
Hiseq x, by Illumina (1 mentions)
Purelink pro 96 viral rna dna purification kit, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Nanophotometer n50, by Implen (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
E coli bl21 de3 competent cells, by Vazyme (1 mentions)
Gel imaging system, by Bio-Techne (1 mentions)
Avaii, by New England Biolabs (1 mentions)
6 protocols using primer star max
1
Generating Translational Reporter for MSL10
2
Amplification and Sequencing of HBV a Determinant
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The “a” determinant region of HBV was amplified by the PCR using PrimerSTAR MAX (TAKARA) under the following PCR conditions: 98℃ 2 min; 98℃ 10 s, 55℃ 5 s and 72℃ 5 s for 35 cycles; 72℃ 5 min. The primers were Af2 (5′-TCCAGGAACA TCAACTACCAG-3′; nt 484–505) and Ar2 (5′-AGGGTTCAAATGTATACCCAA-3′; nt 840–820). The 150 bp paired-end libraries were sequenced by Illumina Hiseq X. The details of NGS workflow are shown in online supplementary figure S1 . The target reads of “a” determinant region from libraries are shown in online supplementary table S3 , and there was no significant difference (p=0.529) between the two groups. Three quasispecies characteristics were calculated by self-written perl scripts (provided by Shanghai OE Biotechnology Co.) at nucleotide and amino acid level, respectively. All calculated values are shown in online supplementary 2 .
3
Quantitative Viral DNA Protocol
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Virus DNA was extracted using PureLink™ Pro 96 Viral RNA/DNA Purification Kit (ThermoFisher Scientific, USA) and reverse transcribed into cDNA. The target fragment was obtained through PCR using primers designed in Table 1 and PrimerStarMax (Takara Bio, Japan). The DNA fragment was ligated with pGEM®-T Easy Vector (Promega, China) using T4 DNA ligase (Takara Bio, Japan). The concentration of the constructed plasmid was measured using Nanophotometer-N50 (Implen, Germany), and diluted into eight gradients at a step size of 10 using Tris-EDTA buffer solution. The copy number at each concentration was computed using . Cycle threshold (Ct) values were obtained for each plasmid concentration using real-time PCR. The standard curve was constructed by fitting the data into a linear regression.
4
Molecular Cloning and Expression
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Cinnamic acid and other chemicals were obtained from Aladdin (Shanghai, China). A DNA gel extraction kit, plasmid purification kit, Primer STAR Max and DNA marker were obtained from TAKARA (Japan). Protein markers, restriction endonucleases and T4 DNA ligase were obtained from Thermo Fisher Scientific (USA). M9 Minimal Salts (M9 buffer) were obtained from Sangon Biotech (Shanghai, China). E. coli RARE(DE3) was purchased from Addgene (USA).
5
Bacterial Protein Expression Protocol
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2-FMA and other chemicals were obtained from Aladdin (Shanghai, China). A DNA gel extraction kit, plasmid purification kit, Primer STAR Max and DNA marker were obtained from TAKARA (Japan). Protein markers and T4 DNA ligase were obtained from Thermo Fisher Scientific (United States). M9 Minimal Salts (M9 buffer) were obtained from Sangon Biotech (Shanghai, China). E. coli BL21 (DE3) competent cells were purchased from Vazyme (Nanjing, China).
6
CAPS Marker Development for BnAHAS1 Mutation
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The sequence comparison of BnAHASs revealed that the nucleotide sequence 540-544 bps from the translation starting site in the WT allele of BnAHAS1 in the line ZS9 was GGTCC, which was mutated to GGTCT in the mutant line K5 (see Section 2.5 ). Therefore, the restriction endonuclease AvaII with the restriction sites GGWCC was employed to develop the CAPS marker for the detection of the causal point mutation in BnAHAS1. A pair of AS-PCR primers (BnA1F1 and BnA1R4, Table S3 ) was designed to amplify the target sequence containing the mutant locus. The PCR mixture (40 μL) contained 100 ng template DNA, 0.75 μM for each primer and 20 μL Primer Star Max (Takara, China). PCR amplification was carried out with 36 cycles at 98 °C for 10 s, 56 °C for 5 s and 72 °C for 10 s. All PCR products were digested with 10 U AvaII (New England Biolabs, Ipswich, MA, USA) for about 30 min at 37 °C at a final volume 25 μL. Subsequently, the products were separated on 2.5% agarose gel, stained with ethidium bromide and visualized using a gel imaging system (Alpha Innotech, Shanghai, China).
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