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. Author manuscript; available in PMC: 2014 Dec 24.
Published in final edited form as: J Virol Methods. 2011 Jul 18;177(1):1–9. doi: 10.1016/j.jviromet.2011.06.019
Problem Solution
Slow Growing 293T This is often resulting from high passage number. Try to
use 293T cells that are below passage 12.
DNA Precipitation If the DNA is not pure it may cause excessive precipitation
during the transfection set up. The Nucleobond Xtra Maxi
EF kit is recommended for endotoxin removal, and
thorough rinsing of the DNA precipitate with 70% ethanol
can remove excess salt.
Cell Clumping If the trypsinization step was insufficient to dissociate the
cells from one another, it is recommended to rinse again
with trypsin to improve recovery.
Uneven Cell Plating This may occur if the incubator shelf is not level.
Cells Peeling Off This can occur for several reasons: too many cells were
plated, or the medium changes were performed too
vigorously, or the HYPERflask was repeatedly knocked.
Column Integrity Fail This is very rare but when it does occur the column must
be replaced.
Flow Path Leak Check all the connections throughout the flow path as this
is usually caused by a single loose connection.
Blocked filter If too much particulate matter or protein (serum) is in the
LV-containing medium then the filter may start to block.
Reduce the protein content in the LV-containing medium
by using serum-free medium. If serum-free medium was
used and the permeate flow seems slow then try closing
the permeate to increase the internal pressure in the filter
to try unblocking some of the pores in the membrane.
High inlet pressure This often occurs as the filter is beginning to clog. Reduce
the back pressure (if additional back pressure has been
applied) and reduce the flow rate.
Aggregation If too much protein is removed then there is a substantial
increase in aggregation. Try to keep some protein present
in the diafiltration mix and avoid diafiltrating in pure
DPBS.
Overheating FP2 can heat up during use, threatening the stability of the
vector. As a precaution, keep the reservoir tube on ice
during the second concentration stage.