Q and A
Frequently asked questions:
- How much transfer vector plasmid do I need to provide to core?
Lenti: is produced in a 15 cm plate and requires 40ug of transfer vector per plate (in the case of a QuickTest prep, the lentivector is produced in a 10cm dish and requires 17ug of transfer vector plasmid per plate).
Retro: is produced in a 10 cm plate and requires 10ug of transfer vector per plate.
- Can you rush my order, I have a deadline?
Unfortunately everyone has deadlines, however, we can occasionally accommodate such a request when it does not interfere with the time lines of the others who have ordered. It depends on our work load.
- Why does it take so long too get viral vector produced?
Is it really that long given the following information. Firstly, we guarantee that the virus order is fulfilled within 4 weeks of receiving the plasmid prep from you. However we generally initiate lentiviral vector production on Mondays (every Monday morning). Therefore if you provide your plasmid on Monday late in the afternoon, it will sit with us in safe storage for 1 week before we start with it. Then production takes approximately 2 weeks. This includes the packaging cell preparation and expansion (3-4 days), the transfection of packaging plasmids (over night), the discard of initial supernatant containing plasmid and then collection of supernatant over three days (4 days), the concentration by ultracentrifugation (1 day), the titration which includes setup and incubation of cells for 3 days followed by FACS analysis. At this point we are at 3 weeks. The last week (week 4) is so that we have a little bit of flexibility to juggle orders to maximise the total output of vector production by maximising use of the facility and infrastructure.
Summary: So if you want Lentiviral vector produced quicker than 4 weeks, find out from Beata when your order could potentially be produced and be sure to deliver the transfer vector plasmid before Monday morning of your production run. You could get the results in less than 2 weeks.
- I just need a quick analysis of my new construct to see if its working. Can’t you do a quickest?
Yes, we have the QuickTest service in full operation. It allows you to get a quick answer on whether your plasmid is expressing the fluorescent marker gene, it provides you with the plate of vector producing cells so you can collect the supernatant and test for function in your own assays, or you can have us use the supernatant to transduce cells to confirm biological function of the vector. You can usually have the quickest performed within a few days of your order if you coordinate with Beata in advance.
The most commonly forgotten things when filling in the order form (please try to remember to include the following information when you place an order):
- the concentration of the DNA in your sample tube
- is it a Lenti or Retro viral vector
- what is the marker gene, if any (e.g. GFP)
- please also provide a map of the plasmid attached to the order form