Pvsvg g

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PVSVG-G is a laboratory equipment product designed for specialized applications. It serves a core function within the research and scientific community. The details of its precise functionality and intended use are not available in this factual and unbiased description.

Automatically generated - may contain errors

Lab products found in correlation

Pdonr207, by Thermo Fisher Scientific (2 mentions) Pog44 flp recombinase expression vector, by Thermo Fisher Scientific (2 mentions) Gateway recombination cloning system, by Thermo Fisher Scientific (2 mentions) Lr reaction, by Thermo Fisher Scientific (2 mentions) Bp reaction, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Polyfect, by Qiagen (2 mentions) Pspax2, by Addgene (2 mentions)

2 protocols using pvsvg g

Recombinant Protein Production and Cell Line Engineering

Check if the same lab product or an alternative is used in the 5 most similar protocols

For cloning the Gateway recombination cloning system (Invitrogen) was used. p27 mutants were generated by site-directed mutagenesis. In BP reactions (Invitrogen) PCR products were recombined with the pDONR207 (Invitrogen) vector. Entry vectors were recombined with destination vectors carrying the desired tags (LR reactions, Invitrogen). To generate stable cell lines, pTO destination vectors (generated by Stephan Geley) were co-transfected with the Flp-recombinase expression vector pOG44 (Invitrogen). To lentivirally transduce p27^−/− MEFs and MDA-MB-231 cells, the plasmids pHR-SFFV-IR-puroSG (a generous gift from Stephan Geley), pVSVG-G (Invitrogen) and psPAX2 (Addgene, Cambridge, MA, USA) were used. pET3c-p27 (wild-type) was used to produce recombinant untagged human p27. Murine p27 was cloned into pET21eSG (a generous gift from Stephan Geley) for expression in E. coli. Rho binding domain of rhotekin was expressed in E. coli using pGEX-2 T-RBD. For transfection Lipofectamine 2000 (Invitrogen) or PolyFect (Qiagen, Venlo, Netherlands) were used.

Podmirseg S.R., Jäkel H., Ranches G.D., Kullmann M.K., Sohm B., Villunger A., Lindner H, & Hengst L. (2016). Caspases uncouple p27Kip1 from cell cycle regulated degradation and abolish its ability to stimulate cell migration and invasion. Oncogene, 35(35), 4580-4590.

+ Open protocol

+ Expand

Cloning and Expression of p27 and RBD

Check if the same lab product or an alternative is used in the 5 most similar protocols

For cloning the Gateway^® recombination cloning system (Invitrogen) was used. p27 mutants were generated by site-directed mutagenesis. In BP reactions (Invitrogen) PCR products were recombined with the pDONR207 (Invitrogen) vector. Entry vectors were recombined with destination vectors carrying the desired tags (LR reactions, Invitrogen). To generate stable cell lines, pTO destination vectors (generated by Stephan Geley) were co-transfected with the Flp-recombinase expression vector pOG44 (Invitrogen). To lentivirally transduce p27^−/− MEFs and MDA-MB-231 cells, the plasmids pHR-SFFV-IR-puroSG (a generous gift from Stephan Geley), pVSVG-G (Invitrogen) and psPAX2 (Addgene, Cambridge, MA, USA) were used. pET3c-p27 (wt) was used to produce recombinant untagged human p27. Murine p27 was cloned into pET21eSG (a generous gift from Stephan Geley) for expression in E. coli. Rho binding domain of rhotekin was expressed in E. coli using pGEX-2T-RBD. For transfection Lipofectamine^™2000 (Invitrogen) or PolyFect (Qiagen, Venlo, Netherlands) were used.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!