A pre-designed shRNA vector set was purchased from Biosettia with shRNAs designed against SIRT3 (Gene ID 23410; Accession NM_001017524.2) in the pLV-H1-CMV-Green plasmid (Biosettia #SORT-B01). NEB stable competent E. Coli cells (New England BioLabs #C3040) were transformed by heat shock with plasmids obtained from Biosettia. Transformed colonies were picked and expanded under antibiotic selection. Plasmids were prepared from bacterial cultures by Maxiprep (QIAGEN #12165). OptiMEM (GIBCO) and Lipofectamine 2000 (Invitrogen #11668019) were used to transfect 9 μg of pLV-H1-CMV-Green plasmid, 6 μg psPAX2 plasmid (Addgene #12260), and 3 μg pMD2.G plasmid (Addgene #12259) into LentiX 293T packaging cells in antibiotic free medium. pMD2.G and psPAX2 plasmids were generous gifts from Didier Trono. Lentivirus containing media was then collected, filtered and used with polybrene to infect target MDA-MB-231 Parental cells. Transduced cells were selected for using fluorescence associated cell sorting (FACS) to generate stable shRNA cell lines.
Neb stable competent e coli cells
Manufactured by New England Biolabs
NEB stable competent E. Coli cells are a laboratory product designed for the storage and transformation of bacterial cells. These cells are genetically modified to be competent, which means they can readily take up and incorporate foreign DNA, such as plasmids, into their genome. The cells are stored in a stabilized form to maintain their competency over an extended period.
Automatically generated - may contain errors
3 protocols using neb stable competent e coli cells
1
Stable Knockdown of SIRT3 in Breast Cancer Cells
2
Stable Knockdown of SIRT3 in Breast Cancer Cells
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A pre-designed shRNA vector set was purchased from Biosettia with shRNAs designed against SIRT3 (Gene ID 23410; Accession NM_001017524.2) in the pLV-H1-CMV-Green plasmid (Biosettia #SORT-B01). NEB stable competent E. Coli cells (New England BioLabs #C3040) were transformed by heat shock with plasmids obtained from Biosettia. Transformed colonies were picked and expanded under antibiotic selection. Plasmids were prepared from bacterial cultures by Maxiprep (QIAGEN #12165). OptiMEM (GIBCO) and Lipofectamine 2000 (Invitrogen #11668019) were used to transfect 9 μg of pLV-H1-CMV-Green plasmid, 6 μg psPAX2 plasmid (Addgene #12260), and 3 μg pMD2.G plasmid (Addgene #12259) into LentiX 293T packaging cells in antibiotic free medium. pMD2.G and psPAX2 plasmids were generous gifts from Didier Trono. Lentivirus containing media was then collected, filtered and used with polybrene to infect target MDA-MB-231 Parental cells. Transduced cells were selected for using fluorescence associated cell sorting (FACS) to generate stable shRNA cell lines.
3
Cloning ZIKV Genome Mutants
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Previously described pACYC177 vector plasmids containing ZIKV PRVABC59 genome from either the 5′ UTR to nt 3498 (pJW231) or from nt 3109 to the end of the 3′ UTR (pJW232) were used to generate WT, TL.PK, and p.2.5′ ZIKV clones (49 (link)). TL.PK and p.2.5′ mutations were cloned into the pJW232 plasmid with gBlocks (IDT, Boulder, CO, USA) using Gibson assembly. Mutant gBlock inserts and pJW232 vector were linearized and amplified using PCR (Table 1 ). Gibson assembly was performed with the NEB Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) with a vector-to-insert ratio of 1:5. Assembled plasmids were transformed into and isolated from NEB Stable Competent E. Coli cells (New England Biolabs) and amplified with rolling cycle amplification. Mutations were verified with Sanger sequencing (Eton Biosciences, San Diego, CA, USA).
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