Three species of planarians: a clonal strain of freshwater planarian (SSP strain of D. japonica20 (link), Dugesia ryukyuensis (menashi mutant strain)32 (link) and S. mediterranea56 (link)) were used in the present study. They were cultured at 23°C in freshwater. Planarians that were 8 mm in length were used in all experiments. In the lidocaine treatment, animals were placed on two pieces of filter paper on ice to paralyze them, and 5 mg ml−1 lidocaine hydrochloride (Sigma) in 1.5% agarose gel (Takara) was then administered using a sharpened glass capillary under a microscope to block the activity of the visual neurons. The control group was treated with anesthesia just posterior to the eyes. Regarding eyecup removal, after animals were paralyzed on ice, the pigment eyecup was scraped out with a sharpened tungsten needle under a microscope. Behavior assays were performed 1 day after surgery, when visual neurons had healed and the pigment cup had not yet regenerated (Supplementary Fig. 2a ). Control animals were scraped at a position next to the right pigment eyecup. All planarians were maintained and manipulated according to a protocol approved by the Animal Care and Use Committee of Kyoto University and Gakushuin University.
Agarose gel
Manufactured by Takara Bio
Sourced in China, Japan
Agarose gels are a type of laboratory equipment used for the separation and analysis of DNA, RNA, and proteins. They provide a porous matrix through which these biomolecules can be separated based on their size and charge. Agarose gels are commonly used in various molecular biology techniques, such as gel electrophoresis.
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Lab products found in correlation
Lidocaine hydrochloride, by Merck Group (1 mentions)
Genomic dna mini preparation kit with spin column, by Beyotime (1 mentions)
Matrigel, by BD (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Ethylenediaminetetraacetic acid edta, by Thermo Fisher Scientific (1 mentions)
Fibronectin fn, by Merck Group (1 mentions)
Penicillin streptomycin p s, by ScienCell (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Real time polymerase chain reaction rt pcr kit, by Takara Bio (1 mentions)
Trizol plus rna purification kit, by Tiangen Biotech (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Fujifilm (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
Dna clean concentrator, by Zymo Research (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Quick gel extraction kit, by Qiagen (1 mentions)
9 protocols using agarose gel
1
Planarian Vision Impairment Protocol
2
DNA Extraction and Visualization
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DNA was extracted using Genomic DNA Mini Preparation Kit with Spin Column (Beyotime Institute of Biotechnology, Shanghai, China). After treatment, the cells were collected and resuspended in PBS, proteinase-K and lysis buffer were subsequently added, and incubated at 70 °C for 10 min. Subsequently, 100% ethanol was added, and the mixture was transferred to the purifying column to obtain DNA after centrifugation and elution. The pelleted DNA was dissolved in TE buffer, electrophoresed on 1.5% agarose gel (Takara Biotechnology, Dalian, China) pre-stained with 0.5 g/mL ethidium bromide (EB) at 50 V for 2 h. The DNA ladders were finally visualized by a UV light source and documented by photography.
3
Anticancer Effects of BetA on Breast Cancer
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BetA was obtained from Tianjin Zhongxin Pharmaceutical Group Corporation Limited (China; purity >98%). Human breast carcinoma MDA-MB-231 and MDA-MB-468 cell lines were obtained from Foleibao Biological Technology Development Co. Ltd (China).13 (link) HMMECs, endothelial cell medium (ECM), foetal bovine serum (FBS), endothelial cell growth supplement (ECGS) and penicillin/streptomycin (P/S) for cell culture were purchased from ScienCell Research Laboratories (USA). L15, F15 and RPMI 1640 media for cell culture were purchased from Hyclone (USA).
Trypsin (0.25%) and ethylenediaminetetraacetic acid (EDTA; 0.02%) were purchased from Gibco (China), and dimethyl sulfoxide (DMSO) was purchased from Solarbio (China). Matrigel was obtained from BD Biosciences (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for MTT assay was purchased from Solarbio (China). RNA was extracted using TRIzol reagent and TRIzol plus RNA purification kit purchased from Tiangen (China). Agarose gel (1%) and real-time polymerase chain reaction (RT-PCR) kit were obtained from Takara (China). Fibronectin (FN) was obtained from Sigma (USA). Dulbecco’s Phosphate-Buffered Saline (DPBS) was obtained from Hyclone (USA).
Trypsin (0.25%) and ethylenediaminetetraacetic acid (EDTA; 0.02%) were purchased from Gibco (China), and dimethyl sulfoxide (DMSO) was purchased from Solarbio (China). Matrigel was obtained from BD Biosciences (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for MTT assay was purchased from Solarbio (China). RNA was extracted using TRIzol reagent and TRIzol plus RNA purification kit purchased from Tiangen (China). Agarose gel (1%) and real-time polymerase chain reaction (RT-PCR) kit were obtained from Takara (China). Fibronectin (FN) was obtained from Sigma (USA). Dulbecco’s Phosphate-Buffered Saline (DPBS) was obtained from Hyclone (USA).
4
Quantitative RT-PCR of Gene Expression
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RNA was extracted from frozen samples of cells and tissues using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions and the concentrations were quantified spectrophotometrically (NanoDrop ND-100, Nanodrop Tec., Wilmington, DE). DNA was digested with RNase-free DNase I (Wako Pure Chemical), which was then inactivated by incubation at 80°C for 10 min.
Starting from 1 μg of RNA, cDNA was synthesized using ReverTra Ace (TOYOBO, Osaka, Japan) according to the manufacturer's descriptions. Primers were designed by Primer3 software (http://frodo.wi.mit.edu/ ) and tested for specificity with NCBI-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ). Real-time quantitative RT-PCR was performed and monitored using Mx3000P QPCR System (STRATAGENE, La Jolla, California, USA). The PCR master mix was based on SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). In reaction wells, cDNA samples (5 μL for a total volume of 25 μL per reaction) were analyzed for the gene of interest. All data were normalized to an internal standard, β-actin. The PCR products were analyzed by agarose gel (Takara Bio Inc., Otsu, Japan)/ethidium bromide (Sigma-Aldrich) to confirm these predicted lengths. Relative quantity was calculated by the ΔΔCt method. The primer sequences are shown in Table 1 .
Starting from 1 μg of RNA, cDNA was synthesized using ReverTra Ace (TOYOBO, Osaka, Japan) according to the manufacturer's descriptions. Primers were designed by Primer3 software (
5
Quantifying Gene Editing Efficiency
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Cells were collected after 48 h post-transfection for genomic DNA extraction. GFP-positive cells sorted by FACS were lysed directly using Buffer L (Bimake). The genomic region flanking the Cas12a targeting site of each gene was PCR-amplified (Additional file 4 : Table S6), and products were purified using DNA Clean & Concentrator (ZYMO Research) following the manufacturer’s protocol. A total of ~ 200 ng purified PCR amplicons was mixed with 1 μl NEBuffer 2 (NEB) and diluted in ddH2O to 10 μl, then subjected to a re-annealing process to form a heteroduplex according to our previously reported procedure [24 (link)]. After re-annealing, the products were treated with T7EI (NEB) following recommending protocol, and 2.5% agarose gels (Takara) were used for further analysis. Indels were calculated via band intensities based on previously reported method [25 (link)].
6
Isolation and Purification of GFPCreERT2
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SW102 E. coli were streaked onto 50 mg/mL kanamycin (Kam; Sigma) plates at 32 °C. A single colony was picked and inoculated in LB medium with Kam and incubated for 4-6 h. The shuttle vector was purified with an Endofree plasmid maxi kit (Qiagen). The vector was digested with NotI and FseI (New England Biolabs), and electrophoresis was performed with agarose gels (Takara, Osaka, Japan). The large fragment of GFPCreERT2 was extracted from the gel with a quick gel extraction kit (Qiagen).
7
Mitochondrial Genomics of Portunid Crabs
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First, the partial sequences of four P. pelagicus genes (12S rRNA, 16S rRNA, Cytb, and COI) and the complete mitochondrial genomes of three other crab species (Charybdis japonica, Scylla paramamosain, and Portunus trituberculatus) were downloaded from the GenBank database. The four genes were amplified, resequenced, and confirmed. The complete mitochondrial genome of P. pelagicus was then obtained by overlapping PCR and sequencing.
The total PCR volume was 25 µL, which contained 0.4 µM each primer, 0.2 mM each dNTP, 1X PCR buffer, 1.5 mM MgCl 2 , 0.75 U Taq polymerase, and 100 ng template DNA. The thermocycler conditions were as follows: an initial denaturation at 94°C for 4 min; then 37 cycles of 30 s at 94°C, 50 s at a primer-specific annealing temperature, and 50 s at 72°C. Finally, the products were extended for 7 min at 72°C. The amplification products were separated on 1.5% agarose gels (TaKaRa, China), and directly sequenced in both directions using an ABI PRISM ® 3730 Automated DNA Sequencer (PE Corporation). The sequences obtained were edited and assembled by EditSeq and SeqMan (DNASTAR).
The total PCR volume was 25 µL, which contained 0.4 µM each primer, 0.2 mM each dNTP, 1X PCR buffer, 1.5 mM MgCl 2 , 0.75 U Taq polymerase, and 100 ng template DNA. The thermocycler conditions were as follows: an initial denaturation at 94°C for 4 min; then 37 cycles of 30 s at 94°C, 50 s at a primer-specific annealing temperature, and 50 s at 72°C. Finally, the products were extended for 7 min at 72°C. The amplification products were separated on 1.5% agarose gels (TaKaRa, China), and directly sequenced in both directions using an ABI PRISM ® 3730 Automated DNA Sequencer (PE Corporation). The sequences obtained were edited and assembled by EditSeq and SeqMan (DNASTAR).
8
Bacterial Protein Expression and Purification
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All of the restriction enzymes, PfuTaq DNA polymerase, agarose gels, DNA purification kit, and T4 DNA ligase were purchased from Takara Biotechnology Co., Ltd (Dalian, China). A Bacterial Gen DNA kit, and a nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Purification kit were purchased from Cowin Biotech Co., Ltd. (Beijing, China). Isopropyl-β-D-thiogalactopyranoside (IPTG) and o-nitrophenyl-β-galactopyranoside (ONPG) were purchased from Sigma Chemical Company (St. Louis, MO, USA).
9
COI Sequence Amplification Protocol
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The primer pair CFF (5'-TCR ACY AAY CAY AAA GAY ATY GGC AC-3') and CFR (5'-ACT TCW GGG TGR CCR AAG AAT CA-3') were used for PCR amplification of COI sequences. PCR was performed in a 25 μL reaction volume containing 2.0 mM MgCl 2 , 0.2 mM dNTP mix, 0.2 μM each primer, 1U Taq DNA polymerase (TaKaRa, Dalian, China), 1X PCR buffer, approximately 100 ng template DNA, and deionized water. The amplification conditions were: initial denaturation of 5 min at 94°C; 35 cycles of 45 s at 94°C, 50 s at 54°C, and 45 s at 72°C; and a final extension at 72°C for 5 min. The PCR products were separated on 1.5% agarose gels (TaKaRa) and then sequenced in both directions using an ABI3730XL (Applied Biosystems).
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