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Quick ligation protocol

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The Quick Ligation protocol is a rapid and efficient method for joining DNA fragments. It facilitates the formation of recombinant DNA molecules by catalyzing the formation of phosphodiester bonds between the 5' phosphate and 3' hydroxyl groups of the DNA segments.

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Lab products found in correlation

Phusion master mix, by New England Biolabs (2 mentions) Psicheck 2, by Promega (2 mentions) Neb10 beta high efficiency cells, by New England Biolabs (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions) Quicklyse miniprep, by Qiagen (2 mentions) Phusion mastermix pcr protocol, by New England Biolabs (2 mentions) Purelink hipure plasmid dna purification kit, by Thermo Fisher Scientific (2 mentions) Nhei hf, by New England Biolabs (1 mentions) Purelink hipure plasmid kit, by Thermo Fisher Scientific (1 mentions) Viafecttm transfection reagent, by Promega (1 mentions) Nano glo dual luciferase reporter assay system, by Promega (1 mentions)

5 protocols using quick ligation protocol

1

Cloning of miR-137 Target 3'UTR

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Primers were designed approximately 500 bp up- and downstream of the putative miR-137 binding site within the 3′UTR (Supplementary Table 1). If the 3′UTR was shorter, the whole 3′UTR region was cloned. Primers contained an AsiSI and a NotI site. PCR fragments were cloned with the two-step program described in the Phusion-MasterMix protocol (New England Biolabs). For the PCR template, genomic DNA was prepared from mouse embryos using the DNeasy Blood & Tissue Kit (Qiagen). The PCR product was purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and digested with AsiSI and NotI. The vector backbone psiCheck2 (Promega) was digested with AsiSI and NotI and treated with alkaline phosphatase (CIP, New England Biolabs) for 1 hr. Vector and insert were gel-purified. Ligation was performed in the ratio 1:3 (vector: insert) using Quick Ligation protocol (New England Biolabs). 5 μL of the ligation was used to transform NEB10 beta high-efficiency cells (New England Biolabs).
Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.
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2

Plasmid Cloning and Dual-Luciferase Assay

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pNL3.1 (#N1031, Promega) was selected as vector and pGL4.53 (#E5011, Promega) as control. Mutant and Wildtype fragment DNAs were inserted into pNL3.1 vector by Quick Ligation Protocol (M2200, New England Biolabs) using NheI-HF (R3131S, New England Biolabs) and HindII-HF (R3104S, New England Biolabs) according to manufacturer instructions (see Supplementary Table S10 for fragment sequences). Transformation was done using 5 Minute Transformation Protocol (C2987H/C2987I) (New England Biolabs) and plasmids were purified by PureLink™ HiPure Plasmid Kits (K2100, Thermo Fisher Scientific) according to instructions. Transfection was done by ViaFectTM Transfection Reagent (E4981, Promega) according to manufacturer instructions with medium to final volume ratio of 4:1. Cells were assay after 24 hours using Nano-Glo Dual-Luciferase Reporter Assay System (N1610, Promega) according to instructions with CentroXS3 LB960 (Berthold Technology) and measurement time of 1 second for both ONE-Glo and NanoDLR.
Sereewattanawoot S., Suzuki A., Seki M., Sakamoto Y., Kohno T., Sugano S., Tsuchihara K, & Suzuki Y. (2018). Identification of potential regulatory mutations using multi-omics analysis and haplotyping of lung adenocarcinoma cell lines. Scientific Reports, 8, 4926.
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3

Synaptotagmin-1 expression plasmid construction

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Primers were designed to synaptotagmin-1 cDNA (NM_001252342.1) and extended by a BamHI site at the 5′ end and an AsiSI site at the 3′ end (Supplementary Table 1). Syt1 was amplified using the Phusion MasterMix PCR protocol (NEB). The vector FUchW H2KK-T2A-Venus, which contains the T2A cleavage peptide sequence, and the Syt1 PCR amplicon were digested with BamHI and AsiSI, gel-purified, ligated with Quick ligation protocol (NEB) and transformed in NEB 10-beta competent cells. Clones were picked, QuickLyse miniprep (Qiagen) performed and the plasmid send for sequencing. The positive clone was retransformed in Stable-3 cells and plasmid purified with PureLink HiPure Plasmid DNA Purification kit (Invitrogen).
Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.
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4

Synaptotagmin-1 expression plasmid construction

Check if the same lab product or an alternative is used in the 5 most similar protocols
Primers were designed to synaptotagmin-1 cDNA (NM_001252342.1) and extended by a BamHI site at the 5′ end and an AsiSI site at the 3′ end (Supplementary Table 1). Syt1 was amplified using the Phusion MasterMix PCR protocol (NEB). The vector FUchW H2KK-T2A-Venus, which contains the T2A cleavage peptide sequence, and the Syt1 PCR amplicon were digested with BamHI and AsiSI, gel-purified, ligated with Quick ligation protocol (NEB) and transformed in NEB 10-beta competent cells. Clones were picked, QuickLyse miniprep (Qiagen) performed and the plasmid send for sequencing. The positive clone was retransformed in Stable-3 cells and plasmid purified with PureLink HiPure Plasmid DNA Purification kit (Invitrogen).
Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.
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5

Cloning of miR-137 Target 3'UTR

Check if the same lab product or an alternative is used in the 5 most similar protocols
Primers were designed approximately 500 bp up- and downstream of the putative miR-137 binding site within the 3′UTR (Supplementary Table 1). If the 3′UTR was shorter, the whole 3′UTR region was cloned. Primers contained an AsiSI and a NotI site. PCR fragments were cloned with the two-step program described in the Phusion-MasterMix protocol (New England Biolabs). For the PCR template, genomic DNA was prepared from mouse embryos using the DNeasy Blood & Tissue Kit (Qiagen). The PCR product was purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and digested with AsiSI and NotI. The vector backbone psiCheck2 (Promega) was digested with AsiSI and NotI and treated with alkaline phosphatase (CIP, New England Biolabs) for 1 hr. Vector and insert were gel-purified. Ligation was performed in the ratio 1:3 (vector: insert) using Quick Ligation protocol (New England Biolabs). 5 μL of the ligation was used to transform NEB10 beta high-efficiency cells (New England Biolabs).
Siegert S., Seo J., Kwon E.J., Rudenko A., Cho S., Wang W., Flood Z., Martorell A.J., Ericsson M., Mungenast A.E, & Tsai L.H. (2015). The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nature neuroscience, 18(7), 1008-1016.
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