Rice protoplasts preparation and transformation were conducted according to the method of Zhang et al., 2011 (link). The sheaths and stems of 30–40 seedlings were cut into 0.5 mm strips and incubated immediately in 10 mL enzyme solution for 4–5 h in the dark at 25 °C with gentle shaking (60 rpm). The protoplasts were purified and resuspended in 1–2 mL MMG solution at a concentration of 5 × 106 cells mL−1. Plasmids (5–10 μg) prepared by an EndoFree Plasmid Midi Kit (CWBIO, Beijing, China) were used for transfection. For the protein degradation assay, DMSO (mock) or 20 μM MG132 (Millipore), H2O (0 μM), 1, 5, and 25 μM E‐64 (Sigma‐Aldrich) or Leupeptin (Sigma‐Aldrich) were added to the protoplasts 12 or 4 h after transfection and treated for 4 or 12 h. For the time course treatment, 20 μM MG132 and/or 50 μM cycloheximide (CHX, Sigma‐Aldrich) were added to the protoplasts 12 h after transfection and treated for 2 h, 4 h, and 6 h. For pathogen mimic treatment, H2O (0 μM), 0.5, 1, 2, and 5 μM Flg22 (Sangon Biotech) were added to the protoplasts 16 h after transfection and treated for 2 h. Transfection experiments were repeated at least three times.
Endofree plasmid midi kit
Manufactured by CWBIO
Sourced in China
The EndoFree Plasmid Midi Kit is a laboratory product designed for the purification of plasmid DNA from bacterial cultures. It provides a reliable and efficient method for obtaining high-quality plasmid DNA, suitable for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Mg132, by Merck Group (2 mentions)
Flg22, by Sangon (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Beyotime (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonsera (1 mentions)
Ni nta sensors, by Molecular Devices (1 mentions)
Gapdh af7021, by Affinity Biosciences (1 mentions)
P38mapk d13e1, by Cell Signaling Technology (1 mentions)
Ni nta beads 6ff, by Smart-Lifesciences (1 mentions)
Triethylammonium bicarbonate teab, by Merck Group (1 mentions)
Tmt kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Trypsin edta, by Beyotime (1 mentions)
Lipofectamine ltx dna transfection reagent, by Thermo Fisher Scientific (1 mentions)
13 protocols using endofree plasmid midi kit
1
Rice Protoplast Preparation and Transformation
2
Plasmid Isolation from Competent Bacteria
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Competent bacteria and encapsulated plasmid DNA were obtained from Invitrogen. A total of 50 µl bacteria solution, 20 ml LB medium and 40 µl ampicillin sodium were placed in aconical flask and shakenat 37 °C for16 hours. After the LB medium becameturbid, a 10-ml suspension was centrifuged under10,000 rpm for 4 min. Thesupernatant was then discarded, and theplasmid was extracted with an EndoFree Plasmid Midi Kit (Cwbiotech, Beijing, China). The concentration of the plasmid was tested with aspectrophotometer (NV3000C, Thermo Fisher Scientific Inc., Waltham, MA USA).
3
Activation of p38 MAPK Pathway
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Reagents: Dulbecco's modified eagle's medium (DMEM) (Sigma-Aldrich, MO, USA); fetal bovine serum (FBS) (Lonsera, UY, South America); p38 mitogen activated protein kinase (p38 MAPK) (D13E1) (8690, Cell Signaling Technology, MA, USA); phospho-p38 MAPK (P-p38 MAPK) (Thr180/Tyr182) (4631, Cell Signaling Technology, MA, USA); GAPDH (AF7021, Affinity Biosciences, Beijing, China); horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (S0001, Affinity Biosciences, Beijing, China); phosSTOP phosphatase inhibitor (Roche, Basel, Switzerland); GC-rich PCR master mix (Sangon Biotech, Shanghai, China); acetonitrile (Fisher Chemical, Fairlawn, USA); triethylammonium bicarbonate (TEAB) (Sigma-Aldrich, MO, USA); Escherichia coli BL21(DE3) competent cells (Solarbio, Beijing, China); Ni NTA Beads 6FF (Smart-Lifesciences, Jiangsu, China); BCA protein assay kit (Beyotime, Shanghai, China); endofree plasmid midi kit (Cwbiotech, Beijing, China); fastpure gel DNA extraction mini kit (Vazyme Biotech, Jiangsu, China); Ni-NTA sensors (ForteBio, CA, USA); tandem mass tagging (TMT) Kit (Thermo, Waltham, USA). Ethylene diamine tetraacetic acid (EDTA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin-EDTA (TE) were obtained from Beyotime Technology (Shanghai, China).
4
Eukaryotic Vector Transfection in RAW264.7 Macrophages
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Eukaryotic expression vectors were extracted using the EndoFree Plasmid Midi Kit (CW2105S; CWBIO), according to the manufacturer’s instructions. Before transfection, RAW264.7 macrophages were seeded in 2-cm glass-bottom cell culture dishes or 10-cm cell culture dishes for 24 h. Then, RAW264.7 macrophages were transiently transfected with the indicated eukaryotic expression vectors using Lipofectamine LTX DNA Transfection Reagent (15338100; Invitrogen) according to the manufacturer’s instructions. Briefly, endofree plasmid DNA diluted in serum-free medium was mixed with PLUS Reagent. The diluted DNA solution was added to diluted Lipofectamine LTX Reagent and incubated for 5 min at room temperature. The resulting DNA – lipid complex was directly dropped into RAW264.7 and incubated for 24 h at 37°C with 5% CO2.
5
G6PD Plasmid Extraction and Transfection
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The G6PD plasmid was designed and synthesized by Vigene Biosciences and the sequence was 5′-GAGGACTAGTTACTGTAATAGTAATCAATTACGGGG, 3′-CCTCTACAAATGTGGTATGGC. EndoFree Plasmid Midi Kit from CWBIO was chosen for the extraction of G6PD plasmid. After extraction, G6PD plasmid was stored at − 80 °C. For transfection, cells were seeded in PolyHEMA-treated 6-well plates at 65% confluency at first. Then, the plasmid DNA (1 μg) was introduced into the cells using Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY, USA) according to the manufacturer's recommendations.
6
MKL1 Silencing and miR-17-5p Regulation
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According to the manufacturer instructions, Negative control mimic (NC mimic), miR-17-5p mimic (mimic), Negative control inhibitor (NC inhibitor), miR-17-5p inhibitor (inhibitor), Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1) were purchased from RIBOBIO biological. All plasmids were extracted with Endo Free Plasmid Midi Kit (CWBIO, China), all fragments and plasmids were transfected with lipofectamine 3000 (Invitrogen, USA) according to the manufacturer instructions.
7
Targeting CDK9, BNIP3, and PINK1 in Cellular Studies
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CDK9 and BNIP3 siRNA were designed and provided by GenePharma (Shanghai, China). The following siRNA sequences were used:
siRNA-CDK9-315 sense: 5ʹ- GGGAGAUCAAGAUCCUUCATT-3ʹ
siRNA-CDK9-315 antisense: 5ʹ-UGAAGGAUCUUGAUCUCCCTT-3ʹ
siRNA-CDK9-881 sense: 5ʹ- GGCCAAACGUGGACAACUATT-3ʹ
siRNA-CDK9-881 antisense: 5ʹ-UAGUUGUCCACGUUUGGCCTT-3ʹ
siRNA-BNIP3 sense: 5ʹ-GCCUCGGUUUCUAUUUAUATT-3ʹ
siRNA-BNIP3 antisense: 5ʹ-UAUAAAUAGAAACCGAGGCTT-3ʹ
siRNA-PINK1 sense: 5ʹ- GCUAACCUGGAGUGUGAAATT-3ʹ
siRNA-PINK1 antisense: 5ʹ-UUUCACACUCCAGGUUAGCTT-3ʹ
HA-CDK9 (14640; deposited by Matija Peterlin), HA-BNIP3 (100781; deposited by Joseph Gordon), MYC-PINK1 (13314; deposited by Mark Cookson) and mito-Keima (56018; deposited by Michael Davidson) plasmids were obtained from Addgene. An EndoFree Plasmid Midi Kit (CWBIO, CW2015S) was used to extract plasmids, which were stored at 20°C.
For transfection, cells were cultured in 6-well plates at a density of 60% overnight attachment. Fresh medium was added one hour before transfection. Then, the plasmid (3 μg per well) or siRNA (400 pmol per well) was introduced to the cells with Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s instructions. All steps were performed using antibiotic-free medium.
siRNA-CDK9-315 sense: 5ʹ- GGGAGAUCAAGAUCCUUCATT-3ʹ
siRNA-CDK9-315 antisense: 5ʹ-UGAAGGAUCUUGAUCUCCCTT-3ʹ
siRNA-CDK9-881 sense: 5ʹ- GGCCAAACGUGGACAACUATT-3ʹ
siRNA-CDK9-881 antisense: 5ʹ-UAGUUGUCCACGUUUGGCCTT-3ʹ
siRNA-BNIP3 sense: 5ʹ-GCCUCGGUUUCUAUUUAUATT-3ʹ
siRNA-BNIP3 antisense: 5ʹ-UAUAAAUAGAAACCGAGGCTT-3ʹ
siRNA-PINK1 sense: 5ʹ- GCUAACCUGGAGUGUGAAATT-3ʹ
siRNA-PINK1 antisense: 5ʹ-UUUCACACUCCAGGUUAGCTT-3ʹ
HA-CDK9 (14640; deposited by Matija Peterlin), HA-BNIP3 (100781; deposited by Joseph Gordon), MYC-PINK1 (13314; deposited by Mark Cookson) and mito-Keima (56018; deposited by Michael Davidson) plasmids were obtained from Addgene. An EndoFree Plasmid Midi Kit (CWBIO, CW2015S) was used to extract plasmids, which were stored at 20°C.
For transfection, cells were cultured in 6-well plates at a density of 60% overnight attachment. Fresh medium was added one hour before transfection. Then, the plasmid (3 μg per well) or siRNA (400 pmol per well) was introduced to the cells with Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s instructions. All steps were performed using antibiotic-free medium.
8
Gene Knockdown Techniques in Cell Studies
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Negative control siRNA (si-NC), siRNA-ALDOA (si-ALDOA), siRNA-PINK1-AS (si-PINK1-AS), siRNA-MKL-1 (si-MKL-1), Negative control mimic (NC mimic), miR- 34a-5p mimic (mimic), Negative control inhibitor (NC inhibitor), miR-34a-5p inhibitor (inhibitor) were purchased from Ribo Biotechnology Co., Ltd. Primers are listed in Supplementary Table 2 . All plasmids were extracted with EndoFree Plasmid Midi Kit (CWBIO, Beijing, China), and all siRNAs, miRNA mimic, miRNA inhibitor and plasmids were transfected with lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer's instructions.
9
Targeted Gene Regulation Experiments
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Negative control mimic (NC mimic), miR-5100 mimic (mimic), Negative control inhibitor (NC inhibitor), miR-5100 inhibitor (inhibitor) Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1), siRNA-CAAP1 (si-CAAP1) were purchased from RIBOBIO biological. All plasmids were extracted with EndoFree Plasmid Midi Kit (CWBIO), all fragments and plasmids were transfected with lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions.
10
Plasmid Overexpression and Depletion
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The HMMR overexpression plasmid, AURKA overexpression plasmid, corresponding serious depletion mutation plasmid, and empty vector was constructed by IGE BIO (Guangzhou, China). The plasmid was amplified by E. coli and extracted under the instructions of a standard protocol provided by the EndoFree Plasmid Midi Kit (CWBIO, China). For plasmid transfection, Opti-MEM™ medium (Thermo Fisher, USA) and X-tremeGENE™ HP DNA Transfection Reagent (Roche, Switzerland) were applied, and the transfection process was performed as suggested. For the Dual-luciferase reports assay, the procedures are presented in Supplementary file 1.
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