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Granulated cell culture media is an easy-to-use, granular form of dry media that is produced by applying fluid bed granulation technology to cell culture nutrient media manufacturing. The patented process allows production of complete formulations of a variety of serum-free, protein-free, and chemically defined media in a dry format.

AGT offers the benefits of liquid media without the cost, storage, and transportation issues. It is a complete medium that is pH and osmolality pre-adjusted. The granules dissolve instantly for faster media preparation time than conventional dry powder media. AGT media can therefore help reduce total cycle costs, decreasing time involved in raw material planning, procurement, and testing as well as media preparation. These benefits are all realized while maintaining comparable cell growth to liquid media.

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Complete BenchStable media (for example, media supplemented with 10% FBS) should be stored at 4 degrees C.

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Gibco Essential 8 Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Gibco Essential 8 Medium is produced under cGMP and has an optimized formulation and growth factor levels that help ensure maximum cell health, pluripotency, and growth, with minimal variability.

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GlutaMAX-I supplement is an improved cell culture supplement that can be used as a direct substitute for L-glutamine in your cell culture medium. Please go here to read more about the properties of GlutaMAX supplement (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/media-supplements/glutamax-media/glutamax-vs-glutamine.html).

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This could be caused by the following:

- Wrong antibiotic or old media: use fresh media.
- Colonies are too old or too small: Use large white colonies from freshly streaked plates.
- Unstable insert caused by special feature of the gene of interest; for example, direct repeats: Incubate the culture at 30 degrees C for 24 hours instead of 37 degrees C overnight.

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Check out our bacterial growth media chart (https://www.thermofisher.com/us/en/home/life-science/cell-culture/microbiological-culture/bacterial-growth-media.html).

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With a vector containing the lac promoter and the LacZ alpha fragment (for alpha-complementation), blue/white screening can be used as a tool to select for presence of an insert. X-gal is added to the plate as a substrate for the LacZ-encoded beta-galactosidase enzyme and must always be present to see the blue color in the blue/white screening.

In cases where the lacIq repressor is present (either provided by the host cells on an F' episome, i.e. TOP10F', or expressed from the plasmid), it will repress expression of LacZ from the lac promoter and prevent the formation of the blue color with X-gal. This repression can be reversed by adding IPTG to the media in addition to X-gal, which will inhibit the action of lacIq and re-activate expression from the lac promoter.

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For the pouches sold as Cat. No. Q30007, each pouch contains reagents to prepare 500 mL of 10X YNB for Pichia media or 1,000 mL of 10X YNB for S. cerevisiae media (since Pichia media is 2X what is used for S. cerevisiae). Below is the formulation for 1X YNB:

Ammonium sulfate, 5 g
Biotin, 0.002 mg
Calcium pantothenate, 0.4 mg
Folic acid, 0.002 mg
Inositol, 2 mg
Niacin, 0.4 mg
Para-aminobenzoic acid, 0.2 mg
Pyridoxine HCl, 0.4 mg
Riboflavin, 0.2 mg
Thiamine HCl, 0.4 mg
Boric acid, 0.5 mg
Copper sulfate, 0.4 mg
Potassium iodide, 0.1 mg
Ferric chloride.6H2O, 0.2 mg
Manganese sulfate.H2O, 0.4 mg
Sodium molybdate.2H2O, 0.2 mg
Zinc sulfate.H2O, 0.4 mg
Potassium phosphate monobasic, 1 g
Magnesium sulfate, 0.5 g
Sodium chloride, 0.1 g
Calcium chloride, 0.1 mg

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The height of the sponge is ~3-4 mm dry. When the sponge is hydrated, its volume is essentially the same - it doesn't swell and increase in size, in fact it becomes slightly bit smaller as it essentially goes back into a hydrogel consistency, especially if the inoculated sponges are centrifuged briefly (as suggested to force cells more into the interior and also to spread the sponge uniformly over the bottom of the well).

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No other components are required.

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There are several options. Our first recommendation is to use the Invitrogen MycoFluor Mycoplasma Detection Kit.

Mycoplasmas are small, self-replicating prokaryotes (0.3 - 0.8 mm diameter), that lack a cell wall and have the ability to adsorb onto host cells. Mycoplasma is one of the most serious forms of cryptic contamination and its presence is not detected unless appropriate tests are made or until some aspect of cell behavior is noticed to have changed. Between 15 and 50% of cell lines submitted to cell banks are contaminated with mycoplasma. Mycoplasma spreads readily among cell lines via reagents and media, the operator and the work surface.

The presence of mycoplasma may invalidate the results obtained with that culture. The presence of mycoplasma-infected cultures can result in the shut-down of the entire laboratory until the infection can be eliminated, whereupon complete restocking is required. The origin of contamination is usually traced to mycoplasma present in animal (bovine) serum or to human oral mycoplasma transferred by droplet infection during cell culture. The simplest test for the detection of mycoplasma in cultures is the use of a fluorescent dye which binds directly to DNA causing fluorescence (e.g. Hoechst 33258) which can be seen by fluorescence microscopy. Mycoplasma positive cells will show intense fluorescent spots on the plasma membranes or show filaments which may be absorbed onto the cells. Uncontaminated cells show only brightly fluorescent cell nuclei. The technique is rapid (less than 30 minutes), but requires heavy contamination (10E6 mycoplasma/ml) to produce a clear positive result. If however, the suspect cells are co-incubated for 2-4 days with an "indicator" cell line (such as 3T3) which is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased. Microbiological culture techniques are available that operate at a greater sensitivity, but it can take up to 21 days to obtain a result, a positive control is needed, and the result may require expert interpretation.

A variety of PCR-based methods are available, some of which have been utilized as commercially available detection kits. It is recommended to use a combination of DNA staining and a PCR-based method once every 3 months for all growing cultures in the laboratory and for every new cell line as it enters the laboratory. In addition, all Master and Working Cell Banks should be tested at the time of freezing. Quality control and good working practice will reduce potential problems. It is important that frozen stocks are created immediately after testing and re-tested before distribution. If cells are cultured for more than 3 months after testing, they should be re-tested. Regulatory bodies now insist that cell cultures used for the production of reagents for diagnostic kits or therapeutic agents are free from mycoplasma infection. Also, some scientific journals have the policy of requiring statements from authors that the culture work reported in those journals is carried out with mycoplasma-free cells. Normally, when contamination with mycoplasma is apparent, the recommendation would be to discard the cultures and start again. If necessary, and only if the contamination is not extensive, then it is often possible to rescue the cells by treatment with one of the commercially available antibiotics. This must only be considered for a remedial action, not as a routine supplement to growth media (and thereby a substitute for good cell culture practice).

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Almost all Gibco cell culture basal media products can be customized using the Gibco Media Configurator. To check on a particular product, use the Media Configurator page (https://www.thermofisher.com/us/en/home/life-science/bioproduction/custom-cell-culture-media-and-services/gibco-custom-media-configurator.html to search by Gibco catalog number, or browse our offering by description. For the customization of non-media Gibco products, such as buffers and growth factors, contact us at custommedia@lifetech.com.

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The CO2 requirement is dependent on the media. Our serum-free 293 media require 8% CO2 to best buffer the media.

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