The bisulfite reaction was carried out according to the protocol provided by Cells-to-CpG™ Bisulfite Conversion Kit (Thermo, Shanghai, China). The converted DNA was amplified by the polymerase chain reaction (PCR) using GoTaq® Hot Start Green Master Mix (Promega, Madison, WI, USA) with BDNF promoter I (product size, 446 bps) forward primer: 5′-AGTTTTTGTAGGATGAGGAAGTGGT-3′ and reverse primer: 5′-ACCATCATAACTAAAAATCTCCAACC-3′. For BDNF promoter IV (product size, 406 bps), the forward primer was 5′-AGTTTGTTGGGGTTGGAAGTG-3′, the reverse primer was 5′-ATACCC(A/G)ATATATACTCCTTCTATTCTACA-3′. PCR amplifications were performed in 30 μL reaction mixtures containing pooled 2 μL of bisulfite-treated genomic DNA, under the following reaction conditions: 5 min at 95 °C, 10 cycles for 30 s at 94 °C, 30 s at 60 °C down 50 °C and 30 s at 72 °C, 25 cycles for 30 s at 94 °C, 30 s at 50 °C and 30 s at 72 °C and finally at 60 °C for 30 min. The PCR products were separated on 2% agarose gels and purified by gel extraction using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA), then cloned into a pCRII vector using TA Cloning Kit Dual Promoter (Invitrogen, Grand Island, NY, USA). Plasmids DNA from 10 colonies were prepared using TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China) and sequenced.
Ta cloning kit dual promoter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TA Cloning Kit Dual Promoter is a laboratory tool designed for the cloning of PCR products. It features a linearized vector with single 3' deoxythymidine (T) overhangs, allowing for direct insertion of Taq polymerase-amplified PCR fragments. The kit includes the cloning vector, competent cells, and other necessary reagents for the cloning process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (5 mentions)
Superscript 2 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Tianprep mini plasmid kit, by Tiangen Biotech (2 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (2 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Cells to cpg bisulfite conversion kit, by Thermo Fisher Scientific (1 mentions)
Gotaq hot start green master mix, by Promega (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
First strand cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqgold, by Thermo Fisher Scientific (1 mentions)
Epitect bisulphite kit, by Qiagen (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Gc rich solution, by Roche (1 mentions)
Advantage 2 polymerase mix kit, by Takara Bio (1 mentions)
Expanded long template pcr system, by Roche (1 mentions)
20 protocols using ta cloning kit dual promoter
1
Bisulfite Conversion and BDNF Promoter Analysis
2
Transcriptome-Guided Cloning and Sequencing
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Total RNA was extracted (TRIzol, Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA (SuperScriptII first-strand synthesis system for RT-PCR, Invitrogen). Gene fragments were isolated by means of RT-PCR with gene-specific primers (Additional file 1 : Table S1) based on sequenced embryonic transcriptomes. All gene fragments were cloned into pCRII vectors (TA cloning kit dual promoter, Invitrogen) and sequenced by a commercial sequencing service (Macrogen, Seoul, South Korea). Newly recovered sequences are available under accession nos. LT560250 (Ek-hbn), LT560249 (Ek-FoxQ2), LT560252 (Ek-nkx2.1/scro), LT560251 (Ek-rx), LT560253 (Ek-vsx/chx), LT560255 (Gm-hbn), LT560254 (Gm-FoxQ2), LT560256 (Gm-nkx2.1/scro).
3
Bisulfate Sequencing of Genomic DNA
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Genomic DNA was first treated by bisulfate according to the DNA Bisulfate Conversion Kit protocol (TIANGEN). BSP was performed using methylation-specific primers and PCR Kit (Tiangen). PCR amplifications were performed in 30 uL reaction mixtures containing 2 uL of bisulfite-treated genomic DNA, and the reaction conditions were 5 min at 95°C, 10 cycles for 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, 25 cycles for 30 s at 94°C, 30 s at 50°C and 30 s at 72°C, and finally at 60°C for 30 min. The PCR products were separated on 2% agarose gels and cloned into a pCRII vector using TA Cloning Kit, Dual Promoter (Invitrogen). Plasmids DNA from 10 colonies were sequenced. Primers were listed in Table S1 .
4
Cloning of Zebrafish MDGA Orthologs
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Through comparative database searches with the corresponding rat and chicken MDGA sequences we identified and subsequently cloned cDNA sequences of three zebrafish MDGA orthologs, MDGA1, MDGA2A and MDGA2B, respectively. Oligo dT primed cDNA, serving as template for MDGA PCRs, was done using the first strand cDNA kit (Invitrogen, Carlsbad, CA). Total RNA used for reverse transcription was isolated from 5 day old wt fish using the QIAShredder and the RNeasy kit (Qiagen, Hombrechtikon, Switzerland). For polymerase chain reaction (PCR) Taq polymerase (Taq Gold; Applied Biosystems) and sequence-specific oligonucleotide primers were used. Amplified DNA pieces were subcloned into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen, Carlsbad, CA) and subsequently sequenced.
5
Amplification and Sequencing of Hox Genes
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Genes were amplified by means of RT-PCR using gene-specific primers (Supplementary File 3 ) and cDNA reverse transcribed from total RNA of a mix of embryonic stages. For all gene fragments, additionally, a nested PCR was performed to boost the PCR results and to apply a higher degree of specificity; for the nested PCR, the first PCR served as a template. We identified two non-overlapping fragments of A. geniculata Antp_B and abdA_B respectively in an embryonic transcriptome (Supplementary File 3 ). Primers for the amplification of these genes were placed in the N-terminal fragment (forward primers) and the C-terminal fragment (backward primers). The complete sequences amplified with these primers, bridging the two non-overlapping fragments of A. geniculata Antp_B and abdA_B, have been deposited in Supplementary File 3 . All gene fragments have been ligated into a pCR-II vector (TA Cloning Kit Dual Promoter, Invitrogen) and sequenced from both directions by a commercially offered sequencing service (Macrogen).
6
Bisulfite Sequencing of Genomic DNA
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Genomic DNA was isolated and treated with sodium bisulphite using the EpiTect Bisulphite Kit (QIAGEN AB, Sollentuna, Sweden) according to the manufacturer's instructions. The bisulphite-converted DNA was amplified by PCR, using the following primers: 5′-TGGGTTAGAGTATAGTTAGGTTAGG-3′ (sense) and: 5′-AATCAAAACCCTCCACATACCTACA-3′ (antisense). The amplification product was electrophoresed on 1.5% agarose gels to confirm the correct product size of 416 base pairs. The product was then extracted from the gel using a QIAquick gel extraction kit (QIAGEN AB, Sollentuna, Sweden), and cloned into a pCR-II vector with the TA Cloning Kit Dual Promoter (Invitrogen/Life Technologies Corp., Foster City, CA, USA) according to the manufacturer's instructions. Ten colonies per cell line were picked for isolation of plasmid DNA, which was then sequenced (Eurofins, Uppsala, Sweden).
7
Molecular Cloning and Sequencing Protocol
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All polymerase chain reaction (PCR) amplifications were performed using the Advantage 2 Polymerase Mix kit (Clontech) following the manufacturer instructions, in some cases including 4% of GC Rich solution (Roche Applied Science) in the master mix. Extra-long PCR (XL-PCR) amplifications were performed using the Expanded Long Template PCR System (Roche Applied Science), following the manufacturer instructions. Reverse transcription PCR (RT-PCR) were performed using either the ThermosTable rTh Reverse Transcriptase RNA PCR kit from Perking Elmer or the One®Step RT-PCR kit (QIAGEN) following the manufacturer’s instructions. The PCR, XL-PCR or RT-PCR products were purified using the Qiaquick PCR purification kit from QIAGEN and cloned either into pSTBlue-1 Vector Vector using the Perfectly Blunt Cloning kit (Novagen) or into the pCR®II Vector using the TA Cloning® Kit Dual Promoter (pCR®II) with One®Shot INVαF’ kit from Invitrogen, as recommended by the manufacturers. Plasmids were isolated and purified using the QIAprep Spin Miniprep Kit (QIAGEN) and sequenced in both strands using the ABI 310 automated DNA sequencer and dye terminator chemistry (Big Dye V3 105 Dye Terminator Sequencing Kit, Applied Biosystems).
8
Cloning and Sequencing of Axud1 cDNA
9
Embryonic Transcriptome Analysis in Spiders
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All animals used in this study originate from the Göttingen strain of P. tepidariorum. The embryos were staged after the embryonic staging table published by Mittmann and Wolff (2012 (link)). Total RNA was extracted from a mix of all embryonic stages using TRIzol® (Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from total RNA with the SMARTer™ PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). Gene-specific cDNA fragments were amplified with primers (see Table S1 ) designed with Primer3 (Untergasser et al. 2012 (link)) and cloned into the pCR®II vector using the TA Cloning® Kit Dual Promoter (Invitrogen, Life Technologies, Carlsbad, CA, USA), with the exception of exd-2, which had previously been cloned with the same degenerate primers as exd-1.
10
In Situ Hybridization of eaat2 Gene
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Cloning of the eaat2 genes into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen) is described elsewhere (Gesemann et al., 2010a (link)). Plasmids containing the genes were linearized for SP6 and T7 in vitro transcription and purified with phenol-chloroform. Digoxigenin (DIG)-labeled antisense RNA probes were generated using DIG-RNA-labeling kit (Roche Diagnostics). Larval whole-mount and adult retina in situ hybridization was done on 5-d postfertilization (5-dpf) larvae and adult retinal cross sections. Detailed protocol of in situ hybridization is described by Huang et al. (2012) (link). Briefly, the tissue was treated with proteinase K and postfixed with 4% paraformaldehyde (PFA) before prehybridization at 64°C. Hybridization of RNA probes was done at 64°C overnight. On day 2, after several stringency washes at 64°C, probes were blocked in 1× Roche blocking solution in Tris/NaCl/Tween. Anti-DIG AP antibody was applied overnight at 4°C. On day 3, after several washing steps, signal was detected by incubation in staining buffer. Stained embryos/retinal sections were fixed with PFA and imaged in glycerol (whole-mount) with an Olympus BX61 light microscope. Images were processed and assembled using Adobe Photoshop and Adobe Illustrator CS5.
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