How much virus do I need to send to OD260 for the virus amplification service?
Please send us either at least 2.5 109 purified virus particles, or at least 1 mL crude virus lysate.
How do I prepare the crude virus lysate that I will send to OD260 for virus amplification?
Follow these steps:
- Seed a 6-cm tissue culture dish with 293 cells at 30% confluence and incubate for 2 days at 37 ºC.
- Infect the newly confluent cell monolayer with your virus.
- Inspect the cells under the microscope daily. Look for cytopathic effect: infected cells become rounded and detach from the dish.
- Harvest when at least 80% of the cell monolayer is detached from the dish. Transfer cells + medium into a 15-mL sterile conical tube.
- Freeze-thaw the infected cell suspension 3 times.
- Clarify the infected cell lysate by a short centrifugation (300 g for 1 min).
- Aliquot the crude virus lysate into 1-mL screw-cap vials and freeze at -70 ºC.
How do I ship virus samples to OD260 Inc?
Shipping adenovirus samples must be done on dry ice using an overnight courier and according to the rules of dangerous goods shipping. The person responsible for shipment must be trained. Most likely your company or institution has already someone prepared for this. If not, training courses can be taken online.
- Place the vial containing your adenovirus sample in a secondary container with absorbent paper.
- Place the secondary container in a styrofoam container with dry ice (5 lbs dry ice per transit day).
- IMPORTANT: in order to protect the virus from inactivation by acidification caused by the dry ice, either the primary container or the secondary container must be placed in a CO2-resistant heat-sealed heavy-duty plastic bag (such as Kapak #400-24).
- Place the styrofoam container in a cardboard box.
- Label the package:
- If the virus is infectious (such as wild-type Ad5), the outer package must be labeled as UN3373 (Biological substance, category B) and UN1845 (dry ice).
- If the virus is not infectious (such as a replication-deficient adenovirus negative for RCA), the outer package must be labeled as UN1845 (dry ice).
Which formulation do you use for purified adenovirus vectors?
We use GTS buffer (2.5 % glycerol (w/v), 25 mM NaCl in 20 mM Tris, pH 8.0) (Hoganson et al, 2002. Bioprocessing J. 1:43-48). The buffer provides stability for at least 4 freeze/thaw cycles, and for at least a year when it is frozen at -80 °C (Gilbert et al, 2004. Mol Ther 9, S294–S294).
What is your address for overnight courier delivery?
800 E. Citation Crt, Suite B
Boise, ID 83716
Do you offer discounts for adenovirus services?
10% discounts are offered when 2 constructs are ordered simultaneously. 20% discounts are offered when 3 or more constructs are ordered simultaneously.
Which facilities are required to work with adenovirus vectors?
The National Institute of Health has designated adenovirus as Level 2 biological agent. For most applications, working with adenovirus requires therefore a Biosafety Level 2 (BL2) facility. The NIH guidelines for research involving recombinant DNA molecules stipulate also that experiments which are likely to either enhance the pathogenicity (e.g. insertion of a host oncogene) or to extend the host range (e.g. introduction of novel control elements) of viral vectors under conditions that permit a productive infection should be performed in BL3 facilities.
A BL2 laboratory should contain:
- A warning sign on the entrance door limiting the access to authorized persons only. The sign should identify the agent, list the name and phone number of the lab director or other responsible person, and indicate any special requirement for entering the lab.
- A Class II biological safety cabinet. A Class II cabinet is a ventilated cabinet for personnel and product protection having an open front with inward airflow for personnel protection, and a HEPA filtered mass recirculated air flow for product protection. The face velocity of the inward flow of air through the full-width open front is 75 feet per minute or greater.
- At least one tissue culture incubator dedicated to infected cell cultures. Another separate incubator is desirable for growing uninfected cells.
- The minimal equipment to handle adenovirus culture without exiting the BL2 lab (such as centrifuges, microscope…).
- A sink for hand washing
- A chemical disinfectant kit or at least a gallon of bleach available for spills
For more information about guidelines, visit this NIH web page.
Which precautions should I take while working with adenovirus?
Work with adenovirus must be performed in a BL2 lab. There you must:
- Always wear a lab coat while in the virus lab. Before exiting the laboratory for non-laboratory areas (e.g. cafeteria, library, administrative offices…), remove your lab coat and leave it in the laboratory.
- Avoid skin contamination with the virus. Always wear gloves (one pair OK, two pairs better for added protection). Once your gloves have been in contact with infectious material, do not touch common appliances such as telephone or doors handles. Change your gloves frequently.
- Keep the lab doors closed while work is in progress.
- Use mechanical pipetting devices. Do not pipet by mouth.
- Decontaminate all work surfaces after you finish your work, and immediately after any spill. Spray a 10% bleach solution, wipe and spray again a 70% ethanol solution. For large liquid spills, add directly concentrated bleach to the liquid, leave for at least 5 minutes, and wipe.
- Perform all procedures with infectious particles in the biosafety cabinet to minimize the exposure of personnel to aerosols. Minimize the creation of aerosols by pipetting virus cultures and suspension very gently. Use aerosol-resistant tips for pipetting virus suspensions. Do not conduct work with infectious materials in open vessels on the open bench.
- Use needle-locking syringes or disposable syringe-needle units for the injection or aspiration of infectious fluids. Extreme care should be used to avoid auto-inoculation and aerosol generation. Needles should not be bent, sheared, replaced in their sheath or guard or removed from the syringe following use. The needle and syringe should be decontaminated by pipetting in and out concentrated bleach a few times and then promptly placed in a puncture-resistant container.
- Decontaminate all contaminated liquid or solid wastes before disposal. Before starting your virus work, pour some bleach into a beaker. Rinse all materials (tissue culture dishes, pipets, tips…) that came into contact with adenovirus with 10% bleach inside the hood before discarding them in the Biohazard trash and autoclaving. Place all materials to be decontaminated off-site in a durable leakproof container which is closed before removal. If possible, leave the contaminated materials in contact with bleach for a few hours before autoclaving (e.g. after rinsing your pipets with concentrated bleach inside the hood, soak them in a cylinder containing 10% bleach before autoclaving).
- Do not leave the BL2 laboratory with live viruses, unless they are in a sealed tube. Cell cultures transduced with adenoviruses should be inactivated either chemically or biochemically before leaving the BL2 facility.
- Store your adenovirus preparations at –70 °C in closed containers labeled with Biohazard warning signs.
- Wash your hands when exiting the laboratory.