CRISPR/Cas9-pP1C.7 vectors were purchased from Nanjing Jirui Biotechnology Co., Ltd. pH-PABE-7-esgRNA is a gift from Caixia Gao (Addgene plasmid # 115620; http://n2t.net/addgene:115620 ; RRID:Addgene_115620). Phanta Max Super ¯Fidelity DNA Polymerase, Taq DNA polymerase, dNTPs, DNA gel purification kit, and quality particle test kit were purchased from Vazyme Biotech Co., Ltd. Restriction enzymes (EcoRI, XbaI, BsaI) were purchased from New England Biolabs (NEB). In-Fusion HD Cloning kits were purchased from Bazaar Medical Technology Co., Ltd. (TaKaRa). The vector transformation strain Ecoli TOP10 was purchased from Biomed Biotechnology Co., Ltd.
Dntps
Manufactured by Vazyme
DNTPs are the basic building blocks used for DNA synthesis. They provide the nucleotides necessary for the enzymatic replication and amplification of DNA.
Automatically generated - may contain errors
Lab products found in correlation
Dna gel purification kit, by Vazyme (1 mentions)
Taq dna polymerase, by Vazyme (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (1 mentions)
Rnase h2 enzyme, by Integrated DNA Technologies (1 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnaseout rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Oligo dt primer, by Vazyme (1 mentions)
Cfx96 touch system, by Bio-Rad (1 mentions)
4 protocols using dntps
1
CRISPR/Cas9 Plasmid Construction
2
LP-LAMP Reaction Mixture Composition
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The reaction mixture of LP-LAMP consisted of 1.6 µM FIP (forward inner) primers and BIP (backward inner) primers, 0.2 µM F3 (outer forward) and B3 (outer backward) primers, 0.8 µM of LF (loop forward) primers, 0.4 µM probe, 8 U of Bst 2.0 DNA polymerase, 6 mM MgSO4, 1 × buffer (New England Biolabs Inc.), 5 mU of RNase H2 enzyme (catalog no. 11-02-12-01, Integrated DNA Technologies), and 1.6 µM of dNTPs (Vazyme). Then, 1 µL of the DNA sample was added, and the final volume was adjusted to 25 µL with dd-H2O. Before closing the lid, we applied mineral oil to the surface to prevent contamination. Each reaction mixture was incubated at 63°C for 60 minutes in a Roche Light Cycler 480 system.
3
Reverse Transcription of Single-Cell RNA
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One hundred thousand cells or nuclei (7 µl) were added with 3 µl 25 µM RT primers (containing RT PolyT Primer (NEB, S1327S), or RT random Primer (Thermo Scientific, SO142), or their mix)), incubated for 5 min at 55 °C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Then a mix of 40 µl RT reaction buffer, containing 10 µl 5 × Reverse Transcription Buffer, 2.5 µl of 100 mM DTT (Thermo Fisher Scientific, P2325), 2.5 µl of 10 mM dNTPs (Vazyme, P031-01), 2.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific, 10,777,019), 3.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific, EP0753), and 21.5 µl nuclease-free water were added. The reverse transcription was incubated as follows (with a heated lid set to 60 °C): 50 °C for 10 min, 3 cycles of [8 °C for 12 s, 15 °C for 45 s, 20 °C for 45 s, 30 °C for 30 s, 42 °C for 2 min, 50 °C for 3 min], 50 °C for 5 min, stored at 4 °C.
4
Quantifying NSCLC-associated miRNAs
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A total of 13 NSCLC-associated miRNAs were assessed using RT-qPCR. TRIzol® kit (Thermo Fisher Scientific, Inc.) was applied to isolate total RNA from A549 cells. Moloney's murine leukemiavirus reverse transcriptase (cat. no. 2641A; Takara Biotechnology Co., Ltd.) was used to perform reverse-transcription reaction using Oligo(dT) primers (Vazyme Biotech Co., Ltd.) and dNTPs (cat. no. 4035; Takara Bio, Inc.). The temperature protocol for reverse transcription was 70˚C for 5 min, followed by ice bath for 2 min and then 42˚C for 60 min. The specific reverse transcription primers for miR-20a were synthesized as previously described (21 (link)). qPCR was performed using TB Green® Premix Ex Taq™ (cat. no. RR420L; Takara Bio, Inc.) on a real-time PCR system (CFX96 Touch system; Bio-Rad Laboratories, Inc.). The thermocycling protocol for qPCR was 95˚C for 10 min, followed by 35 cycles of 95˚C for 15 sec and 58˚C for 20 sec. The expression of OPG mRNA and 13 miRNAs was normalized to those of β-actin and U6, respectively. The primer sequences involved were listed in Table II . The qPCR results were analyzed and calculated using the 2-∆∆Cq method (22 (link)).
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