What is the mode of action on the following antibiotics: Blasticidin, Geneticin® (G418), Hygromycin, and Zeocin™?

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Blasticidin: Nucleoside Inhibits protein synthesis in prokaryotic and eukaryotic cells by interfering with peptidyl transfer reaction of protein synthesis, causing early termination of translation.

Geneticin® (G418): Aminoglycoside Blocks protein synthesis in mammalian cells by interfering with ribosomal function.

Hygromycin: Aminocyclitol Inhibits protein synthesis by disrupting translocation and promoting mistranslation.

Zeocin™: Intercalates with DNA and cleaves it.

Answer Id: 3693

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If there are no zeocin-containing YPD plates readily available, would it be possible to spread zeocin on top of YPD plates and still retain efficient selection of yeast?

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Zeocin™ reagent can be spread on top of YPD plates for selection of yeast if necessary. There is a report that this works well when done with 10-15 3mm glass beads. However, it is recommended that some optimization is performed since top-spreading may dilute the antibiotics' effectiveness.

Answer Id: 3694

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4b26dc4663ccf960c8538d595d0a1d3a_FAQ

In contrast to Geneticin® (G418)-induced cell death, cells treated with Zeocin™ do not always detach and float when they die. Is this typical?

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It is true that a percentage of non-resistant mammalian cells do not round-up from the plate upon Zeocin™ selection as would be seen with G418 or Hygromycin selection. However, one should see some very characteristic morphological changes occurring in those cells that are not resistant. These cells that stick to the culture dish typically display a vast increase in size. This could be best described as being similar to the effects of cytomegalovirus infecting permissive cells. The shape of these cells may also change; taking on an "alien" shape. On close examination of the non-resistant cells, the researcher should observe a distinct breakdown of both the nuclear and plasma membranes. Even though the "cells" are still attached to the plate, they should have the appearance of many holes in these membranes. Also, before the breakdown of the membranes, one can observe open areas in the cytoplasm of the cells that appear to be large, empty vesicles. Although not confirmed, this may be explained by a breakdown of the endoplasmic reticulum and Golgi apparatus, or other scaffolding proteins. Eventually, these "cells" will completely breakdown so that only "strings" of protein are left.

In contrast, Zeocin™ resistant cells should continue to divide at a regular interval to form distinct clumps of cells, or colonies. There should not be a distinct change in morphology, which can be compared to cells not under selection with Zeocin™. It is these colonies of actively dividing cells that contain the resistance gene and are expressing it actively.

If there is concern about the dead cells sticking to the plate, one may do the following to eliminate them: Treat the plate for a couple of minutes with trypsin/versene. Both the healthy resistant cells and the dead cells will dislodge from the plate. The cells can then be replated (without Zeocin™ selection) and the healthy cells will attach again while the dead ones will not. After a couple of hours when the healthy cells have attached to the substrate again, Zeocin™ can be added back to the medium.

Answer Id: 3699

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What safety precautions should be taken when autoclaving Zeocin™-containing media and plates before disposal? Are there any long-term effects for people who are exposed to Zeocin™ on a daily basis?

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Zeocin™, in media or other liquid form, is a health hazard when it comes in contact with your skin or upon ingestion. When it is in the powder solid form, it can become a respiratory hazard as well as contact and ingestion hazard. If the liquid or media is placed in an autoclavable bag, it can be autoclaved. The elevated temperatures within the autoclave will inactivate Zeocin™. Sodium hypochloride (bleach) will also inactivate Zeocin™. We highly recommend wearing the appropriate personal protective equipment (lab coat, gloves, and eye protection) when handling Zeocin™.

Answer Id: 3700

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f92586a25bb3145facd64ab20fd554ff_FAQ

What are the recommended concentrations of antibiotics to use for selection in prokaryotes and eukaryotes?

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For best results, optimal concentrations for selection should be determined empirically in each unique experiment through dose response curves. However, to get a general idea of concentrations that have worked for individual cell types, please click on the following url: http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/selection.html or type in “Selection Antibiotics” into our main search on www.lifetechnologies.com.

Answer Id: 3701

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b181eaa49f5924e16c772dcb718fcd0f_FAQ

What is the molecular weight of Zeocin™?

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MW=1,535. Molecular formula: C60H89N21O21S3.

Answer Id: 3702

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a928731e103dfc64c0027fa84709689e_FAQ

I received Zeocin™ and it looks blue. Is this normal?

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Zeocin™ is a formulation of phleomycin D1, a basic, water-soluble, copper-chelated glycopeptide isolated from Streptomyces verticillus. The presence of copper gives the solution its blue color. This copper-chelated form is inactive. When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu1+ and removed by sulfhydryl compounds in the cell. Upon removal of the copper, Zeocin™ is activated and will bind DNA and cleave it, causing cell death.

Answer Id: 3912

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27b587bbe83aecf9a98c8fe6ab48cacc_FAQ

Which of Invitrogen™'s antibiotics (Geneticin®, Zeocin™, Hygromycin, and Blasticidin) can be used together for selection in mammalian cells?

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All of Invitrogen™'s antibiotics (Geneticin, Zeocin™, Hygromycin, and Blasticidin) can be used together. However, kill curves will need to be determined for each combination of drugs since sensitivity to a given drug tends to increase when combined with other drugs.
Note: Puromycin is of the same family as Blasticidin so Puromycin and Blasticidin may not be compatible.

Answer Id: 3948

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6df182582740607da754e4515b70e32d_FAQ

When doing Pichia expression from a plasmid containing the Zeocin resistance gene, is it necessary to have zeocin in the expression medium?

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There is no need for maintaining Zeocin™ reagent selection in the Pichia expression media since Pichia pastoris transformants are stable integrants with the gene of interest integrated into the genome.

Answer Id: 3697

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