Cells were transfected with 20 nM siRNA duplexes using INTERFERin (Polyplus Transfection, Nottingham, United Kingdom) according to the manufacturer's instructions and were typically analyzed 72 h posttransfection. When cells were additionally transfected with an siRNA-resistant construct during the knockdown, the DNA transfection was carried out 48 h after siRNA transfection. Cells were incubated for additional 24 h and then they were trypsinized, reseeded onto coverslips or glass-bottom dishes, and assayed after another 24 h. GMAP-210 ON-TARGETplus SMARTpool (pool of four siRNAs; L-012684) and each individual ON-TARGETplus siRNA targeting GMAP-210 were from Dharmacon (Thermo Scientific). The sequences of the siRNA oligonucleotides used in this study are siGMAP 1, GGAGAUAGCAUCAUCAGUA; siGMAP 2, CAAGAACAGUUGAAUGUAG; siGMAP 3, GGACAUUACUAAAGAGUUA; and siGMAP, 4, GGGCAAGACUGGAGAGUUA. Luciferase siRNA (GL2; Eurogentec, Southampton, United Kingdom) was used as negative control. Unless indicated otherwise, the results shown were obtained using siGMAP #2.
Luciferase sirna gl2
Manufactured by Eurogentec
Sourced in United Kingdom
Luciferase siRNA (GL2) is a small interfering RNA (siRNA) designed to target the Luciferase gene from the firefly (Photinus pyralis). It is a tool used in gene silencing experiments to study gene function.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using luciferase sirna gl2
1
siRNA-Mediated Knockdown of GMAP-210
2
Cloning and Manipulation of GMAP-210 Protein
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The sequence encoding full-length human GMAP-210 was amplified by PCR from cDNA provided by Francis Barr (University of Oxford, UK), and inserted in frame between the KpnI and NotI sites of pcDNA5/FRT/TO vector (Life Technologies) for insertion of a Myc-tag at the 3′-end of the sequence. GMAP-210 resistant to siGMAP #2 was generated by introducing six silent mutations by site-directed mutagenesis. HRD1–Myc was a generous gift from Stephen High (The University of Manchester, UK). GMAP-210 ON-TARGETplus human SMARTpool (pool of four siRNAs) or each individual ON-TARGETplus siRNA were from Dharmacon, and their sequences are as follows: siGMAP #1, 5′-GGAGAUAGCAUCAUCAGUA-3′; siGMAP #2, 5′-CAAGAACAGUUGAAUGUAG-3′; siGMAP #3, 5′-GGACAUUACUAAAGAGUUA-3′; and siGMAP #4, 5′-GGGCAAGACUGGAGAGUUA-3′. The siGMAP sequence by the Rios laboratory has been published previously (Ríos et al., 2004 (link)). For GM130 knockdown, the ON-TARGETplus human SMARTpool (Dharmacon) was used. Luciferase siRNA (GL2, Eurogentec, denoted siLuc) was used as a negative control.
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