Research and Development on Lentivectors for:

ADDGENEUNITEC

Dr Patrick SALMON @ Department of Neurosciences - Faculty of Medicine - University of Geneva

Lentiviral Vectors Lab @ UNIGE

Standard Procedures for the NEUFO BSL-2 LAB

·          Access to the laboratory is restricted to users listed on the entrance door.


·          All personnel working in the P2 must be authorized by Patrick SALMON or Jozsef KISS, and must have agreed with these P2 procedures.


·          When entering the lab, check the autoclave. If a sterilization cycle has completed, remove bag, put an orange sticker "INACTIVE" on it (they are stored in the lower cupboard) and put the bag in the green waste bin in the corridor outside the P2.


·          Re-enter the lab, put gloves on, put lab coat on.


·          Check for gloves, ethanol 75%, concentrated bleach and paper towels within reach when you work under the hood.


When working in the laminar flow hood:


·          Unplug cable of front lid, remove front lid and put it in the dedicated rack under the hood


·          Switch on air flow, light and vacuum pump under hood


·          Check for airflow green light before working


·          Check that there is enough room left in the hood waste bin


·          Check that there is enough room left in the waste bottle for aspiration of liquid waste AND aspiration of bleach for neutralization (one fifth of total final volume)


·          Check for plastic bottles for bleach and pipet tips waste


·          Open all flasks, culture dishes, tubes, bottles, pipet tips racks, under the hood


·          All solid waste and plasticware must be discarded in the trash bin UNDER the hood (envelopes of pipettes, of flasks, of dishes and of bottles can be discarded in the floor waste bin with a big red bag)


·          All liquids must be aspirated in the liquid waste bottle


·          DO NOT discard liquid in the solid waste bin!


·          Gloves are disposed of and replaced when overtly contaminated, and removed when work with infectious materials is completed or when the integrity of the glove is compromised.



At the end of your work in the laminar flow hood:


·          Flush vacuum tubing with bleach until all liquid waste is neutralized (final color must be yellow, NOT RED or PINK)


·          If the vacuum bottle is full AND liquid is neutralized for at least 10 minutes, disconnect all tubes from bottle and discard liquid in the sink


·          Put 200-300 ml of fresh concentrated bleach in the bottle and put it back in the hood


·          If the solid waste bin is full, close each bag individually UNDER THE HOOD using clips and put this double bag in the big red bag in floor waste bin. If there is no clips, use a piece of thread from the ball on the pipette server or in the bag drawer.


·          Refill the waste bin in the hood with 2 new small plastic bags


·          Clean work surface of laminar flow hood with EtOH 75% and wipe with paper towels.


·          Close hood with UV lid, plug UV lid cable and swith on UV light.


·          Switch off air flow, light and vacuum pump.



When leaving the P2:



·          If the big red bag in the floor waste bin is full, close it with clip, and put it in the autoclave


·          Replace with a new big red bag


·          Start the autoclave


·          Remove lab coat, then glove

 


Notes:


·          Always use bucket cvers when centrifuging


·          Always use concentrated bleach. Diluted bleach is not stable and may be ineffective


·          Laboratory equipment and work surfaces should be decontaminated with detergent and/or EtOH 75% on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials.


·          Use of sharps is not permitted in the P2.


·          Use of glassware must be avoided and should NEVER be used for storage of hazardous material.


·          In case of broken glassware, get a dedicated container and dispose of it by mechanical means such as a brush and dustpan, tongs, or forceps.


·          Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.


·          Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the P2.