北京索萊寶科技有限公司 張凈花 010-56371206
Zeocin
Catalog no. ? ? ? ? ? ? ? ? ? ? ?Quantity ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?Store at -20°C
R250-01 ? ? ? ? ? ? ? ? ? ? ? 1 g (8 × 1.25 ml)
R250-05 ? ? ? ? ? ? ? ? ? ? ? ?5 g (50 ml)
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 Cu1+
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?.
Note: Basic information on using Zeocin? is described in this insert. For
details including Zeocin? structure and photographs of Zeocin? treated
cells, download the Zeocin? manual from www.invitrogen.com.
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).
Part No. R250.pps
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 -20oC 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 observe
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 37oC for 2-3 hours to let cells
attach. Place the cells at 4oC for 2 hours. Remember to buffer the medium
with HEPES. Return the cells to 37oC.
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
25 μg/ml Zeocin? prevents growth of the E. coli strain, TOP10.
Accessory Products
Media for bacteria and mammalian cells as well as transformation
products (yeast and bacteria) and transfection reagents are available from
Invitrogen. Visit www.invitrogen.com for details.