Aliquoting Viruses
Viruses should aliquoted for long term storage and for ready use during surgery. Viruses should always be stored in the -80 freezer until needed. Thermal cycling or the rising and lowering of temperatures will result in the loss of virus efficacy. Viruses and their use are spelled out in both the BIACUC protocol and the Biosafety protocol.
You must be on the Biosafety protocol to handle any of the viruses used in lab.
Materials
100ul centrifuge tubes
Pipette tips
Sterile Fields
PPE (Face Shield, gloves, gown, etc).
Rescue Spray
Pipettes
Sharpie
Centrifuge tube box
Autoclave bags
Sterile PBS (for dilutions)
Prepping for aliquoting
First and foremost, pipette tips and centrifuge tubes must be autoclaved in autoclave bags, better to do autoclave them in separate bags so that tubes and tips can be easily removed. Autoclave extra tips and tubes (more than you think you will need).
Next surfaces should be sprayed down with rescue spray and be wiped clean.
Next a red biohazard bag should be properly labeled with date, PI name, and biosafety protocol number. And then placed into the green trash can.
After the tips and tubes have been sterilized, put on your PPE and then laydown a sterile field and place the tube box onto the sterile field.
Carefully place out the tubes into the slots in the box. After that be sure you have your pipettes and everything else needed before retrieving the virus tube (normally 100ul) from the -80 freezer.
Aliquot out the virus into the desired volumes 5ul is normally done and recommended. However, virus can be aliquoted into the larger volumes however aliquots should not exceed 25ul to avoid potential loss.
Be sure to aliquot all of the virus out of the initial tube into the centrifuge tubes in one session.
Do not allow anyone to enter the surgery suite while you are aliquoting with out proper PPE. Even if they are going in just to grab something for a few seconds.
In the case of large spill of the virus report it to graduate student, or PI immediately.
Aliquoting
Aliquot out the virus into the desired volumes 5ul is normally done and recommended. However, virus can be aliquoted into the larger volumes however aliquots should not exceed 25ul to avoid potential loss.
Take your time and go slow so as to not spill any virus, always be sure to eject the virus at the bottom of the tubes. If your pipette tip for any reason touches any non-sterile surface discard the pipette tip immediately into the red biohazard bag. It is best to close each tube after you are down aliquoting the virus into it.
Dilution of the virus should be only be done with sterile PBS.
Do not touch any surface with the gloves if any virus has made contact with them. Discard them into the red biohazard bag and put on fresh gloves.
Be sure to aliquot all of the virus out of the initial tube into the centrifuge tubes in one session.
Do not allow anyone to enter the surgery suite while you are aliquoting with out proper PPE. Even if they are going in just to grab something for a few seconds.
In the case of large spill of the virus report it to graduate student, or PI immediately.
After aliquoting is finished, double check to make sure all tube lids are secured. Next take a sharpie and label each and every tube with virus name and if diluted with the dilution ratio.
In addition to labeling tubes a piece of tape must be attached to the box which will hold tubes detailing the virus and level of dilution. (Do not just mark on the box with marker will wear off in the -80 freezer).
Clean up
After the labeled box containing the labeled tubes is placed in the -80 freezer clean up should begin.
For clean up anything and everything that has made contact with the viruses that is disposable including the sterile field, pipette tips, gloves etc. should be discarded into red biohazard bag and securely tied and then placed into the red biohazard box at the end of the hallway in the LAR.
Face shields and working bench space should be sprayed down with Rescue spray left to dry for 5 minutes then wiped clean.
Return pipettes to main lab space.
Any unused tubes or pipette tips can be saved and used again in the future, if they have not made contact with the virus.
Updating Virus log
After the aliquoting and clean up is completed, the virus log must be updated with the color of the tubes, the color of the box and the date it was aliquoted along with the name(s) of those who did the aliquoting.
Created 191030 _ Dylan