The PCR products were then purified using the FastPure EndoFree Plasmid Mini Kit (Vazyme, Nanjing, China) and ligated into a pET-30a vector (Abiowell, Changsha, China) according to the manufacturer’s instructions. Afterward, plasmids were transformed into competent Escherichia coli DH5α competent cells, 100 μL of nonresistant LB solution was added, and the cells were shaken for 2 h in a 37 °C incubator and coated. After incubation in an incubator at 37 °C for 12 h, 20 positive clones of single bacteria were selected and incubated overnight at 37 °C in LB liquid medium containing 100 mg/mL Kana for PCR identification of the bacterial solution; the primers for PCR identification are shown in Table 1 . The bacterial solution was identified as a 10 μL PCR system: 2 × Taq Master Mix 5 μL, 10 μmol/L forward, and reverse primers 0.4 μL each, ddH2O 3.2 μL, and bacterial solution 1 μL. Reaction conditions: denaturation at 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and extension at 72 °C for 1 min. The final extension step was at 72 °C for 5 min. Several of these positive colonies were then purified using the FastPure® Plasmid Mini Kit (Vazyme, Nanjing, China) and sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.
Fastpure plasmid mini kit
Manufactured by Vazyme
Sourced in China
The FastPure Plasmid Mini Kit is a laboratory equipment used for the rapid and efficient extraction of high-quality plasmid DNA from bacterial cultures. The kit utilizes a silica-based adsorption method to purify plasmid DNA, ensuring the removal of contaminants and inhibitors.
Automatically generated - may contain errors
Lab products found in correlation
Fastpure gel dna extraction mini kit, by Vazyme (12 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (4 mentions)
Phanta max master mix, by Vazyme (2 mentions)
Rapid taq master mix, by Vazyme (2 mentions)
Clonexpress ultra one step cloning kit, by Vazyme (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lentiguide puro, by Addgene (1 mentions)
Phanta master mix, by Vazyme (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Yeast plasmid extraction kit, by Solarbio (1 mentions)
Lr clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Bp clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
Pce2 ta blunt zero vector, by Vazyme (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Hiscript 3 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Clonexpress one step cloning kit, by Vazyme (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
High fidelity dna polymerase, by Vazyme (1 mentions)
26 protocols using fastpure plasmid mini kit
1
Cloning and Sequencing of Plasmid DNA
2
CRISPRi Transcription Interference Assay
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Anneals of sgRNA oligos targeting the same SE region were pooled, cloned into lentiGuide-puro (Addgene #52963). Pooled sgRNA plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme, DC201-01) and packaged into lentivirus as previously described. Stable dCas9-KRAB-expressing cells were infected with the pooled-sgRNA lentivirus and qPCR-tested for transcription interference on SE-associated gene. All CRISPRi-sgRNA sequences were listed in supplementary Table 6 .
3
Molecular Cloning Reagents Sourcing
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All restriction enzymes were purchased from New England Biolabs (Beijing, China), 2× Phanta® max master mix, 2× Rapid Taq master mix, ClonExpress® II one step cloning kit, FastPure® Plasmid Mini Kit and FastPure® Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China), peptone and yeast extract were purchased from Thermo Scientific Oxoid Microbiology Products (Basingstoke, England), isoamyl alcohol and n-dodecane were purchased from Aladdin® (Shanghai, China).
4
Cloning and Transfection of PFN1 Variants
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The human PFN1-coding sequence was amplified from human cDNA using 2× Phanta Master Mix (Vazyme, NanJing, China) and cloned into a pLVX-IRES-ZsGreen1 and pEGFP-C3 vector using the ClonExpress II One Step Cloning Kit (Vazyme). The primers were synthesized by TsingKe Biology Company (Beijing, China) and are listed in Supplementary Table S4 . After gene recombination and transformation into Escherichia coli, plasmids were extracted using the FastPure Plasmid Mini Kit (Vazyme). The plasmids were then selected according to sequence screening. All genes were transfected into cells using lentivirus infection. pLVX-EF1α-IRES-puro, pLVX-EF1α-IRES-puro-PFN1-Flag, pLVX-EF1α-IRES-puro-PFN1-R88L-Flag, pLVX-EF1α-IRES-puro-PFN1-H119E-Flag, pLVX-EF1α-IRES-puro-PFN1-H133S-Flag, and pLVX-EF1α-IRES-puro-ROCK2-myc were purchased from General Biology Company (Chuzhou, China). ROCK1 pcDNA3.1-HA-C was purchased from You Bao Biology (Changsha, China). PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in Supplementary Table S4 . Plasmids and siRNAs were transfected into cells using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Waltham, MA, United States) according to the manufacturer’s instructions.
5
Constructing Plasmids with GDH
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All the primers for constructing plasmids are listed in Table 1 . The PCR product of GDH was digested with EcoRI and HindIII and cloned into EcoRI-HindIII-digested pET-28a(+) after agarose gel purification. The ligation mixture was first transformed into chemically competent E. coli DH5α cells. The positive recombinants were further confirmed by PCR. The sequenced plasmid was transferred into competent E. coli Bl-21 cells. GDH mutants were constructed using the overlapping primer method (Sudhir et al., 2016 (link)). The plasmids were constructed using a FastPure Plasmid Mini Kit, FastPure Gel DNA Extraction Mini Kit, and ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). Primer synthesis and nucleic acid sequencing were completed by Sangon Biotech (Zhengzhou, China).
6
Protoplast Transformation Verification
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To verify whether the protoplasts isolated using this method could be used for plasmid transformation, we fused the conventional transient expression vector pCAMBIA1302-CaMV35S::eGFP to the membrane-localized GhPIN1 gene, the CPS4 gene (pCaMV35S::CPS4-YFP), a nuclear localization marker, a membrane-localized marker, or an endoplasmic reticulum (ER) localization marker. Plasmid extractions were performed with the Vazyme FastPure Plasmid Mini Kit (#DC201–01).
7
Heterologous Expression of MsVDAC in N. benthamiana
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MsVDAC was amplified using specific primers (5′-cgggggactcttgacgagctc ATGGCTAACGGTCCAGCAC-3′; the SacI restriction site is underlined) and (5′-catgtcgactctagaggatcc AGGCTTGAGAGAAAGAGCGAG-3′; the BamHI restriction site is underlined). The amplicons were double-digested with SacI and BamHI and then ligated to a pCAMBIA1300-GFP vector. The ligated products were transformed into Escherichia coli strain DH5α competent cells. The plasmids were subsequently extracted from positive clones using a FastPure Plasmid Mini Kit (Vazyme, Nanjing, China) and sequenced. A 35S-MsVDAC-GFP positive recombinant plasmid was introduced into Agrobacterium tumefaciens strain EHA105. Then, the solution was injected into the blade back of Nicotiana benthamiana leaves; after 48 h of culture in the dark, the leaves were observed via a laser scanning confocal microscope.
8
Validation of Yeast Two-Hybrid Screening
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To validate the screened interactions, each of the potential positive prey plasmids was again co-transformed with pGBKT7-P0 into Y2HGold. The detailed steps were as follows. Each screened putative positive prey plasmid was extracted from the corresponding yeast clone by using a Yeast Plasmid Extraction Kit (Solarbio, D1160, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). To separate the probable multi-prey transformants and raise the concentration of plasmids, the obtained plasmids were transformed into E. coli DH5α and cultured on LB (containing 100 µg/mL of ampicillin) agar plates. The positive clones were verified by PCR and followed by plasmid extraction by using the FastPure Plasmid Mini Kit (Vazyme, DC201-01). Subsequently, each inferred positive prey and pGBKT7-P0 bait plasmid were co-transformed into Y2HGold and planted on QDO/X agar plates to demonstrate the interactions. Under these steps of confirmation, the blue colonies indicate true positive interactions.
9
Cloning and Expression of MdYABBYs in Tobacco
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Firstly, specific primers were designed according to the CDS sequence of MdYABBYs by Snapgene 3.2.1, removing the stop codon and adding the XbaI and KpnI restriction sites. Then, the first strand cDNA of M. dodecandrum leaves was used as a DNA template for PCR amplification, and the PCR products were separated by 1.2% agarose gel electrophoresis and purified with a FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China). The purified product was ligated to the pMDC202 vector and transformed into Escherichia coli (DH5α) by a ClonExpress® Ultra One Step Cloning Kit (Vazyme, Nanjing, China). The positive clones were selected and sequenced by Sangon Biotech (Shanghai, China) Co., Ltd. The correct clone was extracted from the plasmids by FastPure Plasmid Mini Kit (Vazyme, Nanjing, China). After that, the plasmid of MdYABBYs was transferred to agrobacterium tumefaciens (GV3101) by the freeze-thaw method. Finally, the 35S::MdYABBYs−GFP vector and the vector without the genes were transferred into tobacco leaf separately. After 48 h, GFP fluorescence signals were observed by a confocal laser scanning microscope (LSM710; CarlZeiss, Jena, Germany). The primers of MdYABBYs are listed in Supplementary Table S1 .
10
Split Luciferase Complementation Assay for RALF and FER
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For split luciferase complementation assay, the whole CDS sequences of CqRALF15 and AtRALF22, as well as the ectodomain of AtFER and CqFER, were amplified by using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). PCR products were purified by FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China) and cloned into pDONR207 ENTRY vector using BP Clonase II Enzyme Mix (Thermo Fisher Scientific). Plasmids were extracted using FastPure Plasmid Mini Kit (Vazyme, Nanjing, China). Finally, all fragments mentioned above were recombined into pCAMBIA-nLUC or pCAMBIA-cLUC vectors using LR Clonase II Enzyme Mix (Thermo Fisher Scientific). For the generation of CqRALF15 transgenic plants in Arabidopsis, CqRALF15 was also recombined into the destination vector pEarleyGate 101. Primers used for constructs are listed in Supplementary Table 11 .
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