[Cell-biology] Tips for Retrovial Transfection

Kevin Carbajal via cellbiol%40net.bio.net (by kevcarby from gmail.com)
Tue Aug 18 13:37:43 EST 2009


I'm not getting any positive cells in my titers.  I'm using 293T,
lipofectamine, and a Nature protocol with the DNA described therin (a GFP
reporter).
(Paper: Retrovirus-mediated single-cell gene knockdout technique in adult
newborn neurons in vivo, Tashiro and Gage).

Pretty standard protocol as far as I know.  I didn't purify the virus by
ultracentrifugation and just did titers with filtered viral sups instead. No
positives by FACS.

Where are the most common pitfalls?  As far as I can tell, my cells are
good, DNA is good, Lipofectamine is expensive, and the protocol was followed
with a small change.


Thank you for any help!
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