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

Product FAQ

Answer

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: E3912

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e9ccd495a2a9c59c50a7b6fa3907fff9_FAQ

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

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Answer

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: E3693

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930bb9e689203845528729313d3a2286_FAQ

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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Answer

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: E3694

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a57abb1dae7e2857e2909b824401f092_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?

Product FAQ

Answer

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: E3699

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9d7a8e2d06bcf2e3a0268fad918c7cc3_FAQ

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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Answer

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: E3700

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566b33b249bab90750d7f8bb548ea611_FAQ

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

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Answer

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: E3701

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b5ff8626d6afae9110e0d47e8142fc4d_FAQ

What is the molecular weight of Zeocin™?

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Answer

MW=1,535. Molecular formula: C60H89N21O21S3.

Answer Id: E3702

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93be181d5f822c8bdef22055c8a4c199_FAQ

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

Product FAQ

Answer

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: E3948

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8e441594d47e26b35930de3e97f92c07_FAQ

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

Product FAQ

Answer

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: E3697

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c5ba1d7e4c4270a02ba8a26796d8b05c_FAQ