What's the allowed vector size for In-Fusion?

In-Fusion technology allows easy cloning of single or multiple DNA fragments directly into large vectors (e.g., adenoviral vectors at 32.6–36 kb, cosmids at 46 kb*) in a single reaction, without intermediate cloning into transfer/shuttle vectors. (Please see Figures 1, 2, 5, and Table III of the Adeno-X Adenoviral System 3 Brochure for details.)
*The 46-kb cosmid vector, assembled by In-Fusion Cloning, was transformed in chemically competent bacteria, allowing the rescue of the recombinant vector. It was not used in the actual cosmid packaging reaction.

In-Fusion Cloning products

Care guide

Caution
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This care sheet provides general information only for handling Carolina™ bacterial cultures. When you work with bacteria, it is imperative that you use sterile techniques at all times. Failing to use sterile techniques can contaminate cultures and work areas, and cause health and safety risks. See our Carolina™ Techniques for Studying Bacteria and Fungi Manual (item #154664) for descriptions of sterile techniques and standard practices for handling bacterial cultures.
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Immediate care and handling
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When your bacterial cultures arrive, immediately open the shipping container and remove the cultures. We ship cultures in tubes, plates, and as MicroKwik Culture® freeze-dried cells. Visually inspect each culture. Ensure that tubes are intact with caps securely in place, plates have no cracks and lids are secured by tape, and plastic bags containing MicroKwik Culture® cells are securely closed with the enclosed tube and vial intact.

You must seal any culture damaged during shipment—and anything it contaminated—in an autoclavable bag, and then sterilize it by autoclaving or soaking all contaminated materials in disinfectant. Note: Never discard a damaged, unsterilized culture in the trash. Know and follow your school or district’s guidelines for proper disposal. Contact our Customer Service at 800.334.5551 for a replacement of your damaged culture. It will help to have your order number available when you call.

Hold most cultures at room temperature, 20 to 22° C (68 to 73° F). For tube cultures, loosen the cap and keep the culture tube upright in a test tube rack or beaker. For best results, use cultures within 3 to 5 days of receipt; however, most bacterial cultures remain usable for up to 4 weeks when held at room temperature. Keep in mind that Carolina provides a variety of bacterial cultures and some have very specific care and handling needs that differ from these general guidelines. Vibrio fischeri, for example, must be kept in the dark and subcultured 2 to 3 times per week to maintain bioluminescence.

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Maintaining and culturing
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Eventually your bacterial culture will deplete the nutrients of the medium in or on which it is growing and will need subculturing. Before working with bacterial cultures, wash your hands with soap and water, ensure that the work area is draft free, and wipe the work surface with 70% alcohol or similar disinfectant. Note: Always check the culture for signs of contamination immediately prior to using. Never work in an area where food is prepared or consumed.

Transfer broth cultures to fresh broth using a sterile pipet or loop or streak onto agar using a sterile inoculating loop. For faster growth, you can incubate most cultures at 25 to 30° C (77 to 86° F). After making the transfers, clean the work area with disinfectant and wash your hands again. Either autoclave the old stock cultures and glassware, or cover them with disinfectant overnight. We recommend the use of Clavies® Autoclavable Bags (item #831642) for disposal.

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Biosafety
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In the US the CDC (Centers for Disease Control) sets standards for the safe handling of microorganisms according to their Biosafety Level (BSL). There are 4 Biosafety Levels with BSL-1 being the lowest risk microorganisms and BSL-4 being the highest risk. Bacterial cultures provided by Carolina Biological Supply Company fall into BSL-1 and BSL-2. Our catalog and online listings identify BSL-2 bacterial cultures as pathogens. They are only available as MicroKwik Culture® freeze-dried cells, and we can only ship them to colleges and universities. Cultures not identified in our listings as pathogens are BSL-1.

The CDC standards for working with BSL-1 microorganisms include the following:
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Follow all standard microbiological practices.
Work can be performed on an open lab bench or table.
Wear personal protective equipment (PPE), i.e., lab coats, gloves, and eye protection, as needed.
A sink for hand washing.
Doors that separate the lab room from the rest of the facility.

For working with BLS-2 microorganisms the CDC recommends all of the above, plus the following:
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Restricted access to the lab room while cultures are being used.
PPE to include face shields as needed.
All procedures that can produce a splash or an aerosol must be performed within a biological safety cabinet.
An autoclave or other method of decontamination for proper disposal.
Self-closing doors that separate the lab room from the rest of the facility.
An eyewash station.

For a fuller discussion of CDC criteria for handling microorganisms see the following link: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf [link].

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FAQs
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Which agar should I use for my bacterial cultures?
In Carolina’s print and online catalogs, the product descriptions for our bacterial cultures include the type of nutrient medium that we use for each culture. This information also appears on the label affixed to the culture. A wide range of bacteria will grow on Nutrient Agar (item #821862) and Tryptic Soy Agar (item #822022).

Can I pour my own plates if I don’t have an autoclave?
See our Nutrient Agar Bottle (item #776360) and other prepared media bottles. A bottle containing 125 mL of medium will pour 4 to 5 standard 100 × 15-mm plates. Watch our “How to Melt and Pour Agar Plates” video (at www.carolina.com) for a demonstration of the technique.
Should I order a tube, plate, or MicroKwik Culture®?
Tube cultures are best for stock. A tube culture is often streaked on a plate and incubated before lab use. Order a plate culture for convenience and immediate use. You can receive a plate culture and use it in a lab on the same day. A MicroKwik Culture® is best used for longer-term storage and to receive a culture of a pathogen. A MicroKwik Culture® can be held at room temperature for up to 2 months before being activated or for 6 to 8 months refrigerated.

Which cultures should I order for antibiotic testing?
Gram-positive and Gram-negative bacteria often give different results when tested against the same antibiotic. For that reason, we recommend testing against Bacillus cereus (item #154872), which is Gram-positive, and Escherichia coli (item #155068), which is Gram-negative. We recommend broth cultures because this makes it easier to spread the culture over the surface of an agar plate. See our Carolina BioKits®: Antibiotic Sensitivity kit (item #154740) for more information.
You recommend holding a culture at room temperature, but the recommended temperature for Escherichia coli (item #155065) is given in your catalog as 37° C. Why is that?
Thirty-seven degrees C is the incubation temperature, the temperature needed for maximum culture growth. The culture is mature when we ship it out and does not need further incubation. Maintaining the culture at room temperature allows you to hold it longer before use.