DNA was extracted from freesia flowers using NuClean Plant Genomic DNA Kit (CWBIO) according to the manufacturer’s instruction. Promoters of FhDFR1/FhDFR2 and FhDFR3 were cloned using Genome Walking Kit (TaKaRa) following the instructions. The -1466 bp of FhDFR1/FhDFR2 and -1132 bp of FhDFR3 from the initiation condon “ATG” were amplified as promoters and cloned into Pst I and Sac I digested AtDFR-pro:GUS construct to generate FhDFR1/FhDFR2-pro:GUS and FhDFR3-pro:GUS, respectively. All the other constructs used for protoplasts transfection have been described previously (Li et al., 2016 (link)). All the plasmids were prepared using the EndoFree Plasmid Maxi Kit (CWBIO) following the manufacturer’s instructions.
Endofree plasmid maxi kit
Manufactured by CWBIO
Sourced in China
The EndoFree Plasmid Maxi Kit is a lab equipment product designed for the purification of endotoxin-free plasmid DNA. It utilizes an anion-exchange resin to capture and purify plasmid DNA from bacterial cell lysates.
Automatically generated - may contain errors
5 protocols using endofree plasmid maxi kit
1
Cloning Freesia Flower Promoters
2
Plasmid Transfection Protocol
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The cDNAs were individually cloned into the pCAG vector. All plasmids used for transfection of mammalian cells were prepared using the EndoFree Plasmid Maxi Kit (CWBiotech).
3
Cloning of Alzheimer's-Related Proteins
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The coding DNA sequences for human PS1 or PS2 and their variants, APH-1aL, PEN-2 and NCT were individually cloned into the pMLink vector25 (link). All plasmids used for transfection of mammalian cells were prepared using the EndoFree Plasmid Maxi Kit (Cwbiotech).
4
Production and Purification of SARS-CoV-2 S1 Protein
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The S1 protein gene sequence was retrieved from NCBI (Spike residues 319–668, GenBank: MN908947.3). The signal peptide encoding sequence of the SARS-CoV-2 spike protein was employed in the S1 protein constructs to facilitate secretion. The S1 protein plasmid was synthesized by Sangon Biotech (Shanghai, China). The products were ligated into the pcDNA3.1 (+) vector and subsequently introduced into E.coli. DH5α competent cells through transformation. The recombinants were amplified and abstracted utilizing the EndoFree Plasmid Maxi Kit (CWBIO; CW2104). The plasmid was transiently transfected into HEK293F using PEI (Yeasen Biotechnology, China) [70 ]. The cell supernatant was collected and filtered to identify protein 72h after transfection. Then proteins were purified by using an immunoaffinity chromatography column based on the SARS-CoV-2 Spike protein-specific mAb preserved in our laboratory. The eluted fractions were pooled and exchanged to 0.01M PBS pH 7.4 via Sephadex G-25 (GE Healthcare, USA).
5
Generation of Induced Pluripotent Stem Cells
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Lv-teto-Oct4, Sox2, Klf4, and c-MYC were kindly provided by Dr. Rudolf Jaenisch's laboratory at the Whitehead Institute for Biomedical Research. The plasmid preparation and iPSC induction procedure were performed according to a previously reported method (Kang et al., 2009) . Plasmids were extracted with the EndoFree Plasmid Maxi Kit (Cwbio, China). Then, 293T cells were transfected with the plasmids for Lv-teto-Oct4, Sox2, Klf4, and c-MYC along with the lentivirus packaging plasmids ps-PAX-2 and pMD2G (VigoFect, Vigorous Biotechnology, China). The medium containing virus was collected 48 hours after transfection. A total of 2 3 10 4 MEFs were seeded and infected with virus-containing medium supplemented with 4 mg/mL polybrene for 12 hours. Infected MEFs were cultured in ES medium supplemented with 1 mg/mL doxycycline hyclate for approximately 14 days. Colonies were cultured in ES medium in the absence of doxycycline for 2-3 days before being mechanically picked. iPSC lines were cultured at 37 C with 5% CO 2 .
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