Sample RNA was extracted and reversely transcribed as per instructions of reverse transcription kit (Promega, USA). Firstly, reverse transcription reaction was carried out using 4 μL RNA as a template and the loading machine was ABI9700 (ABI, USA). The reaction solution included: 4 μL 5 × RT buffer, 0.4 μL random primers, 1 μL MMLV (U/μL), 0.5 μL 10 mM dNTPs, 10.1 μL diethylpyrocarbonate (DEPC)-treated water (Sangon Biotech, China), and 4 μL RNA template. The conditions set for reaction were 37°C for 1 h and 95°C for 3 min. The reaction system included the following: PCR mix buffer 2 × 10 μL, 1 μL upstream primer and 1 μL downstream primer, 4 μL of template, and 6 μL of DEPC- treated water. The reaction was carried out at 93°C for 3 min, 93°C for 30 s, 55°C for 45 sec, with a total of 40 cycles, then 72°C for 5 min, and 4°C forever. The applied primers included: GATA6-F, 5’- GGATTCTTGGTGTGCTCTGG-3’; GATA6-R, 5’-ATTTTTGCTGCCATCTGGAC-3’; BMP2-F, 5’-TCTTCCGGGAACAGATACAGG-3’; BMP2-R, 5’-TCTCCTCTAAATGGGCCACTT-5’; MGP-F, 5’- TGAAGAGCCTGATCCTTCTTGCCA-3’; MGP-R, 5’- TAGAGCGTTCTCGGATCCTCTCTT-3’; β-actin-F, 5’-GATGCTCCCCGGGCTGTATT-3’; and β-actin-R, 5’-GGGGTA CTTCAGGGTCAGGA-3’.
Diethylpyrocarbonate depc treated water
Manufactured by Sangon
Sourced in China
Diethylpyrocarbonate (DEPC)-treated water is a specialized laboratory solution that has been treated with DEPC, an RNase inhibitor. This product is commonly used to prepare water for various molecular biology applications where the presence of RNase enzymes needs to be minimized.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcription kit, by Promega (1 mentions)
Lba cas12a, by New England Biolabs (1 mentions)
Mgcl2, by New England Biolabs (1 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
Dynabeads myone streptavidin t1, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by New England Biolabs (1 mentions)
Nah2po4, by Sinopharm (1 mentions)
Na2hpo4, by Sinopharm (1 mentions)
Real time pcr system, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Gdna eraser kit, by Takara Bio (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Bstni, by New England Biolabs (1 mentions)
Dna marker, by Sangon (1 mentions)
Nebuffer 3, by New England Biolabs (1 mentions)
Bst dna polymerase large fragment, by Vazyme (1 mentions)
Nt bstnbi, by New England Biolabs (1 mentions)
Dna oligonucleotides, by Sangon (1 mentions)
6 protocols using diethylpyrocarbonate depc treated water
1
RNA Extraction and RT-qPCR Analysis
2
Sensitive Viral Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chloroauric acid (HAuCl4), trisodium citrate, Na2HPO4, NaCl and NaH2PO4 are obtained from Sinopharm Chemical Reagent Co., Ltd. Streptavidin-modified magnetic bead (Dynabeads™ MyOne™ Streptavidin T1, 10 mg/mL) is purchased from Thermo Fisher Scientific. All HPLC-purified DNA oligonucleotides and crRNA are purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the oligonucleotides are listed in Tables S2-S4 . The diethylpyrocarbonate (DEPC)-treated water were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Lba Cas12a and Buffer 3.1 (pH 7.9) containing NaCl (100 mM), Tris-HCl (50 mM), MgCl2 (10 mM), DTT (1 mM), were purchased from New England Biolabs (Beijing, China). RNase inhibitors were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The RNase-free environment throughout the experiments using DEPC- treated water and RNase-free tips and tubes. GX/P2V beta coronavirus isolated by using Vero E6 cells were provided from Yigang Tong’s laboratory.
3
Quantitative Analysis of miR-499a-5p and PPM1D Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the OS tissues and normal cells transfected with either miR-499a-5p or its NC using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then l mg total RNA was reverse transcribed using a PrimeScript RT Reagent Kit and a gDNA Eraser kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. Sequences of all primers were as follows: miR-499a-5p forward, 5′-ATGTAGCGTGCGACCG-3′ and reverse, 5′-CAGGCTGACGCACTCTGTGCT-3′; U6 forward, 5′-CCATCGGAAGCTCGTATACGAAATT-3′ and reverse, 5′-GGCCTCTCGAACTTGCGTGTCAG-3′; PPM1D forward, 5′-TTCAGAGCTCCATAACAAGC-3′ and reverse, 5′-GGACCTATCGCATCCGACCG-3′; and β-actin forward, 5′-GCTGTCCCTGTATGCCTCT-3′ and reverse, 5′-TGTCACGCACGATTTCC-3′. Subsequently, 1 µl miR-499a-5p or PPM1D primers (Sangon Biotech Co., Ltd.), 10 µl SYBRGreen (Takara Biotechnology Co., Ltd.), 6 µl diethylpyrocarbonate (DEPC)-treated water (Sangon Biotech Co., Ltd.) and 2 µl cDNA, with a total volume of 20 µl, was then processed in a Real-Time PCR system (Roche Diagnostics, Basel, Switzerland). The following thermocycling conditions were used: 5 sec at 95°C, followed by 40 cycles of 1 sec at 95°C and 20 sec at 65°C. The comparative 2−ΔΔCq method was used for relative quantification of gene expression (16 (link)). Each sample was analyzed ≥3 times.
4
CRISPR-Cas12a-based SARS-CoV-2 Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bst DNA polymerase (Large Fragment) derived from Bacillus stearothermophilus and 10 × thermoPol buffer were obtained from Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China). Nt.BstNBI, BstNI, and 10 × NEBuffer 3.1 were purchased from New England Biolabs (NEB). LbCas12a (cpf1) was provided by Tolo Biotech Co., Ltd (Shanghai, China). Diethyl pyrocarbonate (DEPC)-treated water, deoxynucleotide triphosphates (dNTPs), 4S GelRed, DNA Marker, and all the DNA oligonucleotides were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China). SARS-CoV-2 RNA transcribed in vitro standard material was provided by National Institute of Metrology, China, while the gene composition and quantitative concentration were shown in Table S3 . SARS-CoV-2 RNA, MERS-CoV RNA, SARS-CoV RNA, and crRNA were synthesized by Gene Pharma (Shanghai, China) and purified by HPLC. The corresponding sequences of the above-mentioned oligonucleotides were displayed in supporting information (SI, Tables S1 and S2 ).
5
RT-LAMP Assay for Gene Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ThermoPol Reaction Buffer Pack, AMV Reverse Transcriptase and its reaction buffer, Bst 2.0 DNA Polymerase, HiScribe™ T7 High Yield RNA Synthesis Kit, Monarch RNA Cleanup Kit, were all purchased from New England Biolabs (Beijing, China). dNTP Set 100 mM Solutions, RNase-Free DNase I and Eco32 I were obtained from Thermo Scientific (Shanghai, China). TRNzol Universal Reagent, EndoFree Mini Plasmid Kit II and Universal DNA Purification Kit were acquired from TIANGEN (Bei-jing, China). TB Green Premix Ex Taq was bought from Takara Biotechnology (Dalian, China). HiScript II Q RT SuperMix for qPCR was purchased from Vazyme (Nanjing, China). Betaine was supplied by Sigma (USA). Diethyl pyrocarbonate (DEPC) treated water, oligonucleotides and pcDNA 3.1 recombinant plasmids containing the DNA fragment of target genes were acquired from Sangon Bio-technology (Shanghai, China). The sequences of oligonucleotides are listed in Table S1 . The real-time fluorescence measurements of the RT-LAMP reactions were performed using an Applied Biosystem 7500 Fast real-time PCR instrument.
6
Optimized Cell Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zinc chloride, anhydrous, 99.95% (metals basis) was purchased from Alfa (Beijing, China). All of the synthetic oligonucleotide sequences used in this study were synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China) and purified using HPLC. The sequence information is listed in Table S1 (ESI †). Diethyl pyrocarbonate (DEPC)-treated water, 1 Â TAE electrophoresis buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0-8.4) and agarose were obtained from Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Hoechst 33342 and 10 mM phosphate buffered saline (PBS, pH 7.2-7.4) were acquired from Solarbio (Beijing, China), Cell Counting Kit-8 (CCK-8 reagent) and 10 mM HEPES buffer (100 mM NaCl, 20 mM MgCl 2 , pH 7.2) were purchased from Beijing BioDee Biotechnology Co., Ltd (Beijing, China). Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) solution (containing 0.02% EDTA) and penicillin-streptomycin were obtained from Gibco (USA). Methanol was obtained from Beijing Chemical Co. (Beijing, China). The HepG2 cells and CCC-HEL-1 cells were obtained from Peking Union Medical College Hospital. All reagents were purchased and used without further purification, deionized water obtained from a Milli-Q water purification system (18.2 MO cm À1 , Milli-Q, Millipore) was utilized in all the experiments.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!