Five mg of lyophilised Amolops wuyiensis skin secretion was dissolved in 1 mL of Lysis/Binding buffer. A Dynabeads® mRNA DIRECT™ Kit (Dynal Biotech, Merseyside, UK) was used for mRNA isolation. The cDNA library construction was performed using Clontech SMARTer® RACE 5′/3′ Kit (Takara Bio, USA, Inc., San Jose, CA, USA). A nested universal primer (NUP) and a degenerate sense primer (S1: 5′-GAWYYAYYHRAGCCYAAADATG-3′; W = A + T, Y = C + T, H = A + C + T, R = A + G, D = A + G + T) were used. The cDNA ends were rapidly amplified by a PCR thermal cycling system with repeated denaturation, annealing, and extension. The products were analysed by gel electrophoresis and then purified using a Hi-Bind DNA mini-column (Omega Bio-Tek, Norcross, GA, USA). The process of DNA ligation was performed using a pGEM-T Easy Vector System (Promega Corporation, Madison, WI, USA). After that, the recombinant plasmid DNA was cloned in JM109 high-efficiency competent cells (Promega, Madison, WI, USA). A Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for sequencing reaction and the products were analysed using an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).
Clontech smarter race 5 3 kit
Manufactured by Takara Bio
Sourced in United States
The Clontech SMARTer® RACE 5′/3′ Kit is a laboratory equipment product designed for rapid amplification of cDNA ends. It provides a method for generating full-length cDNA sequences from limited amounts of RNA.
Automatically generated - may contain errors
Lab products found in correlation
Dual glo luciferase assay system, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Jm109 high efficiency competent cells, by Promega (1 mentions)
Abi 3730 automated sequencer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Hi bind dna mini column, by Omega Bio-Tek (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using clontech smarter race 5 3 kit
1
Amolops wuyiensis Skin Secretion cDNA Library Construction
2
Characterization of BgElo12 Promoter Activity
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The 5′ end of BgElo12 was obtained by 5′ Rapid Amplification of cDNA Ends (RACE). The 5′ RACE cDNA library was prepared using Clontech SMARTer RACE 5’/3’ Kit (Takara) according to the user manual with the gene-specific primer (S4 Table ) and kit-provided Universal long primer. The amplified fragments were cloned into pRACE vector and sequenced. About a 2.7-kb sequence upstream of BgElo12 was amplified and cloned into pGL3-basic vector, and the CDS sequences of BgDsxM and GFP (control) were separately cloned into the expression vector pCDNA3.1. The HEK293T cells were cultured in a 24-well plate with 500 μL of Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) for 24 hours before transfection, and the restructured pGL3-basic vector (200 ng/well) was co-transfected with the expression vectors (200 ng/well) to HEK293T cells using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA). The pRL-TK that encoded a Renilla luciferase was also co-transfected as an internal control. The transfected cells were cultured at 37°C for 36 hours and subjected to luciferase activity analysis using the Dual-Glo Luciferase Assay System (Promega).
3
Cloning and Transfection of BgElo12
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The 5' end of BgElo12 was obtained by 5' Rapid Amplification of cDNA Ends (RACE). 5' RACE cDNA library was prepared using Clontech SMARTer RACE 5'/3' Kit (Takara) according to the user manual with the gene-specific primer (S4 Table ) and kit-provided Universal long primer. The amplified fragments were cloned into pRACE vector and sequenced. About a 2.7 kb sequence upstream of BgElo12 was amplified and cloned into pGL3-basic vector, and the CDS sequences of BgDsx M and GFP (control) were separately cloned into the expression vector pCDNA3.1. The HEK293T cells were cultured in a 24-well plate with 500 μL of DMEM medium (Thermo Fisher Scientific) for 24 h before transfection, and the restructured pGL3basic vector (200 ng/well) was co-transfected with the expression vectors (200 ng/well) to HEK293T cells using Lipofectamine TM 3000 (Invitrogen, Carlsbad, CA, USA). The pRL-TK that encoded a Renilla luciferase was also co-transfected as an internal control. The transfected cells were cultured at 37℃ for 36 h (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. and subjected to luciferase activity analysis using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA).
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