博来霉素/博莱霉素/Zeocin

博来霉素/博莱霉素/Zeocin  博来霉素/博莱霉素/Zeocin 
产品编号：A3270
产品名称：博来霉素/博莱霉素/Zeocin 
英文名称 Zeocin
浓度 100mg/ml
外观（性状） 淡绿色液体
储存条件 -20℃
有效期 2年
规格 1.25ml 8*1.25ml
单位 瓶
说明: Zeocin是一种糖蛋白抗生素， 在体内能作用于大多数细菌(包括E. coli)、真菌(如：酵母菌)、植物细胞、动物细胞。
Zeocin试剂是博来霉素抗生素家族的一个成员。 其抗性由编码一种 13,665 道尔顿蛋白质的 Sh ble 基因赋予。 在表达这种蛋白质的细胞中，Zeocin无法结合和切割细胞 DNA。 Zeocin对大多数好氧细胞有效，可用于选择哺乳动物和昆虫细胞系、酵母以及细菌。 选择细胞时，Zeocin的用量为 50-2000 ug/ml（通常 300 ug/ml），具体取决于细胞类型。
Zeocin
Quantity 0.125 g (8 × 1.25 ml)
Store at -20°C
Description
Zeocin is a formulation of phleomycin D1, a basic, water-soluble, copperchelated glycopeptide isolated from
Streptomyces verticillus and shows strong toxicity against bacteria, fungi (including yeast), plants, and mammalian cell
lines. The blue color of the solution is due to the presence of copper and the copper-chelated form of Zeocin is inactive.
When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu+ and removed by sulfhydryl compounds
in the cell. Upon copper removal, Zeocin is activated, and binds and cleaves DNA, causing cell death. A Zeocin resistance
protein of 13,665 Da, has been isolated and characterized. The protein is the product of the Sh ble gene
(Streptoalloteichus hindustanus bleomycin gene), binds stoichiometrically to Zeocin and inhibits its DNA strand cleavage
activity. Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin.
Specifications
Contents: 100 mg/ml solution in deionized, autoclaved water.
Shipping/Storage: Shipped on blue ice. Store at -20°C.
E. coli Selection: 25-50 μg/ml in low salt LB medium (NaCl concentration should not exceed 5 g/liter.)
Yeast Selection: 50-300 μg/ml in YPD or minimal medium
Mammalian Cells 50-1000 μg/ml in suitable medium (varies with cell Selection: line).
Handling Zeocin
• Always wear gloves, a laboratory coat, and safety glasses when handling Zeocin containing solutions.
• Zeocin is light sensitive. Store the antibiotic and plates or medium containing the antibiotic in the dark.
• Reduce the salt in bacterial medium and adjust the pH to 7.5 to keep Zeocin active as high ionic strength and acidity or
basicity inhibit Zeocin activity.
• Store Zeocin at -20℃ and thaw on ice before use.
Zeocin Selection in E. coli
Host: Must not contain the Tn5 transposon (i.e. TOP10, DH5, DH10).
Medium: Use Low Salt LB Medium (10 g Tryptone, 5 g NaCl, and 5 g Yeast Extract) at pH 7.5 to prevent inactivation of
Zeocin.
Selection: Use 25-50 μg/ml of Zeocin for selection in E. coli.
Zeocin Selection in Yeast
Yeast: Saccharomyces cerevisiae, Pichia pastoris
Medium: YPD with 1 M sorbitol (electroporated cells); YPD or minimal plates (chemically transformed cells). Test the
medium adjusted to pH values ranging from 6.5-8.0 and select the pH that allows you to use lowest
Zeocin concentration.
Transformation Method: Use electroporation, lithium cation protocols, or EasyComp Kits. Do not use spheroplasting for
yeast transformation with Zeocin containing plasmids as it results in complete cell death.
Selection: Use 50-300 μg/ml of Zeocin, depending on the yeast strain, and media pH and ionic strength. Perform a kill
curve to determine the lowest Zeocin concentration required to kill the untransformed host strain.
Note: Allow the cells to recover for 1 hour in YPD medium after transformation. To obtain efficient Zeocin selection,
plate at low cell densities (use 10, 25, 50, 100, and 200 μl of transformation reaction).
Zeocin Selection in Mammalian Cells
Use 50-1000 μg/ml of Zeocin to select stable cell lines (the average is about 250-400 μg/ml). Depending on the cell line,
it takes 2-6 weeks to generate foci with Zeocin. Determine the minimum concentration required to kill your
untransfected host cell line prior to generating stable cell lines (see below).
Determining Zeocin Sensitivity
1. Plate or split a confluent plate to obtain cells at ~25% confluency. Prepare a set of 8 plates. Grow cells for 24 hours.
Remove the medium.
2. Add medium with varying Zeocin concentrations (0, 50, 100, 200, 400, 600, 800, and 1000 μg/ml) to each plate.
3. Replenish selective medium every 3-4 days and obs.erve the percentage of surviving cells. Select the concentration
that kills the majority of cells within 1-2 weeks.
Selecting Stable Integrants
1. Transfect your cell line and plate onto 100 mm culture plates. Include a sample of untransfected cells as a negative
control.
2. After transfection, wash the cells once with 1X PBS and add fresh medium to the cells.
3. Forty-eight to 72 hours after transfection, split the cells using various dilutions into fresh medium containing Zeocin at
the pre-determined concentration required for your cell line. To have a better chance at identifying and selecting foci,
we recommend using different cell dilutions.
4. Feed the cells with selective medium every 3-4 days until cell foci are identified.
5. Pick and transfer colonies to 96- or 48-well plates. Grow cells to near confluence before expanding to larger wells or
plates.
Zeocin Selection in Mammalian Cells, Continued
Selection Tip
If your cells are more resistant to Zeocin, split cells into medium containing Zeocin and incubate the cells at 37℃ for 2-3
hours to let cells attach. Place the cells at 4℃ for 2 hours. Remember to buffer the medium with HEPES. Return the cells
to 37°C.
Incubating the cells at 4°C stops the cell division process for a short time, allowing Zeocin to act, resulting in cell death.
Maintaining Stable Cell Lines
• Maintain cells in the same Zeocin concentration used for selection
• Reduce the Zeocin concentration by half or to a concentration that just prevents growth of sensitive cells but does not
kill them (refer to the kill curve experiment)
Product Qualification
Zeocin is lot qualified by demonstrating that LB media containing 35 μg/ml Zeocin prevents growth of the E. coli strain,
TOP10.
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