The specific sequence used for CDKN2D silencing was selected from the WI siRNA Selection Program (http://sirna.wi.mit.edu/ ). A negative control sequence was used (AGC GUG UAG CUA GCA GAG G). A total of 2 × 106 NB4 cells were suspended in 100 μl Amaxa Nucleofector Solution V and mixed with 3 μg siRNA. Cells were then transfected by electroporation using Amaxa Nucleofector II device and program X-001. Six hours after transfection, cells were cultured for another 48 h in the absence or presence of 1 μM ATRA. The siRNA sequence targeting CDKN2D was as follows: forward: 5′- GUACCAGUCCAGUCCAUGAUU-3′ reverse: 5′- UCAUGGACUGGACUGGUACUU-3′.
Amaxa nucleofector solution 5
Manufactured by Lonza
Sourced in Switzerland
The Amaxa Nucleofector solution V is a specialized buffer designed for use with the Amaxa Nucleofector system, a laboratory instrument for the transfection of cells. The solution provides the necessary components to facilitate the efficient introduction of nucleic acids, such as DNA or RNA, into a variety of cell types.
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Lab products found in correlation
5 protocols using amaxa nucleofector solution 5
1
CDKN2D Silencing in NB4 Cells
2
Transfection of Sarcoma Cell Lines
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GIST-T1, GIST430, RMS176, LMS04 and EWS502 cell monolayers were disaggregated with trypsin and resuspended in Amaxa Nucleofector solution V (Amaxa Biosystems) at a concentration of 1 × 106 cells per 100 μl. Nucleofection was performed using program T-030 on a Nucleofector II machine (Amaxa Biosystems). One microgram of pCR3.1-EGFP or pCR3.1-miniDMD plasmid was used for electroporation. Transfected cells were selected with G418 for 5 days before analyses.
3
Transfection of Sarcoma Cell Lines
4
Generation of Ku70 3A Mutant in Mouse Fibroblasts
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Mouse Ku70 cDNA was PCR amplified using the primers mKu70-Bam-ATG: 5′-GGCGGATCCATGTCAGAGTGGGAGTCCTAC-3′ and mKu70-Stop-Xho: 5′-GGCCTCGAGTCAGTTCTTCTCCAAGTGTCTG-3′ and then subcloned into the pCDNA3.1-YFP-F2 mammalian expression vector using BamHI and XhoI following standard protocols. PCR-directed mutagenesis using complimentary oligonucleotides was then used to generate mouse Ku70 3A. The following primers were used for the mutagenesis: mKu70-307A: 5′-GACTTTTAATGTAAACGCCGGCAGTCTACTCC-3′ and mKu70-314A/316A: 5′-CAGTCTACTCCTGCCTGCTGACGCCAAGCGGTCTCTGAC-3′ with the mKu70-307A primer set used first and then the mKu70-314A/316A primer set. To make Ku70−/− MEFs that stably express wild-type and Ku70 3A, 4 μg of linearized plasmid containing wild-type or Ku70 3A cDNA was transfected using Amaxa nucleofector solution V (Lonza) and program T-020. Cells stably expressing mKu70 were selected using 800 μg/ml hygromycin.
5
TRPML1 and PIKfyve Transfection Protocols
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