tricks to increase retroviral titer?
(by aawara from FEMA-trailer.org)
Mon May 28 11:07:07 EST 2007
In <1180363688.517662.228520 from q75g2000hsh.googlegroups.com>,
Antonio <Antonio.Sarikas from gmail.com> wrote:
> Thanks DK, but what do you pellet? The virus ist secreted into the
> supernatant which I then collect at day 2 and 3 posttransfection.
> Ultracentrifugation of the supernatant might be problematic since the
> retroviral glycocalix is sensitve to shearstress.
DK is correct, you can definitely concentrate retroviral supes by
ultracentrifugation. I did this routinely with retroviruses as a
graduate student, and now my lab does it with amphotropic vectors
and pantropic vectors.
Here's how we do it -
a) Layer 33 ml cell supernatant on 5 ml 20% sucrose in an SW 28 tube.
b) Spin at 26,000 rpm for 2 hours at 5oC. Make sure the rotor, centrifuge
and buckets are pre-chilled (we leave the rotor and buckets in the
c) You will see a pellet at the bottom of the tube. Suck the
supernatant off. Resuspend the pellet in 0.5 ml of PBS (or complete
media - which is what we use).
d) Leave the resuspend pellet O/N at 4oC, and then either freeze at
-80oC or use.
Email: echo 36434455860060025978157675027927670979097959886449930P | dc
More information about the Methods