Pmko 1 gfp

Manufactured by Addgene

Sourced in United States

PMKO.1-GFP is a lab equipment product that serves as a plasmid. It contains a GFP (green fluorescent protein) reporter gene.

Automatically generated - may contain errors

Lab products found in correlation

Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Pspcas9 bb 2a puro v2, by Addgene (1 mentions) Plenti cmv puro dest, by Addgene (1 mentions)

Close spelling variants of this product

PMKO.1‐puro GFP shRNA (3 citations) PMKO-GFP (2 citations) PMKO.1 GFP vector (2 citations)

3 protocols using pmko 1 gfp

Retroviral Transduction of A375 Cells

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pMKO.1-GFP (https://www.addgene.org/10676/) and pMKO.1-shTERT (https://www.addgene.org/10688/) plasmids obtained from Addgene. For the retroviral production, the platinum A cell line (Cell Biolabs) was transfected with pMKO.1-GFP and pMKO.1-shTERT plasmids using the Calcium Phosphate method. 24 hours after transfection, the fresh medium was added. Starting from Day 2 after the initial transfection, viral supernatants were collected for two consecutive days. Viral supernatants were centrifuged at 1200 rpm for 5 minutes and filtered using 0.45 um filters. A375 cells were infected with viral supernatants supplemented with Polybrene at 8µg/ml. Stably infected cells were established by either FACS sorting of GFP positive cells or puromycin selection.

Zhang G., Wu L.W., Mender I., Barzily-Rokni M., Hammond M.R., Ope O., Cheng C., Vasilopoulos T., Randell S., Sadek N., Beroard A., Xiao M., Tian T., Tan J., Saeed U., Sugarman E., Krepler C., Brafford P., Sproesser K., Murugan S., Somasundaram R., Garman B., Wubbenhorst B., Woo J., Yin X., Liu Q., Frederick D.T., Miao B., Xu W., Karakousis G.C., Xu X., Schuchter L.M., Gangadhar T.C., Kwong L.N., Amaravadi R.K., Lu Y., Boland G.M., Wei Z., Nathanson K., Herbig U., Mills G.B., Flaherty K.T., Herlyn M, & Shay J.W. (2018). Induction of Telomere Dysfunction Prolongs Disease Control of Therapy-Resistant Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(19), 4771-4784.

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LincIN shRNA Knockdown Protocol

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For LincIN short hairpin RNAs (shRNAs), sense and antisense oligos were designed using the Whitehead Institute online tool (http://sirna.wi.mit.edu/) and synthesized by Integrated DNA Technologies (IDT) (Coralville, IA, USA). The oligos were then annealed prior to cloning into the Age I and EcoR I restriction enzyme sites of pMKO.1-GFP, which was purchased from Addgene (Cambridge, MA, USA). After screening for LincIN knockdown efficacy, two of the most efficient shRNA constructs were chosen from a total of seven shRNA designs (sequences listed in Additional file 1: Table S1). In addition, the full length of LincIN was synthesized by Genewiz, Inc. (Cambridge, MA, USA), and was cloned into the pRetroX-IRES-ZsGreen vector (Clontech, Mountain View, CA, USA) at the BamH I and Not I restriction enzyme sites.

Jiang Z., Slater C.M., Zhou Y., Devarajan K., Ruth K.J., Li Y., Cai K.Q., Daly M, & Chen X. (2017). LincIN, a novel NF90-binding long non-coding RNA, is overexpressed in advanced breast tumors and involved in metastasis. Breast Cancer Research : BCR, 19, 62.

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Plasmid Construction for Mitochondrial Studies

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Twnk ORF was amplified from plasmids pJet2-Twinkle and pROSA-K320E 67 (link) and cloned in pmCherry-N1. For retroviral vector generation, Twinkle and Twinkle-mCherry, ORF was amplified and subcloned into pLenti-CMV Puro DEST (Addgene #17452). All ORFs were subcloned into pBabe-Puro vector, kindly provided by Dr. Bernhard Schermer, and verified by Sanger sequencing. pLX304-TWINKLE-APEX2 vector was kindly provided by Dr. Alice Ting. To generate Mus musculus Twnk vectors, Homo sapines TWNK was replaced by Twnk ORF and subcloned into pBABE-Puro vector. Mitochondrial matrix APEX2 control vector (Addgene #72480) was also subcloned into pBABE-Puro. For generation of Tfam-GFP, total RNA from mouse liver was isolated and converted into cDNA using TRIzol (Thermo Fisher) and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) using PolyT as feeder. Tfam cDNA was cloned into pEGFP-N1.
Atad3 KD and Samm50 KD clones were generated by transducing MEFs with the vector pMKO1-GFP (Addgene #10676) containing a shRNA (Table M1). Empty vector and a scramble containing vector were used as a control. For CRISPR Cas9 KO generation two gRNA directed to exon 4 and exon 5 (Table M1) were cloned in pSpCas9 (BB)-2A-Puro V2.0 (Addgene #62988).

Pla‐Martín D., Sen A., Kallabis S., Nüchel J., Maliphol K., Hofmann J., Krüger M., & Wiesner R.J. (2021). Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.

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