How to make stable transfectants

Richard J. Dudley rjdudley at YAHOO.COM
Fri Jul 24 12:00:15 EST 1998


Preliminary steps (in no particular order):

1) Grow your cells.  How you do this will depend on the cell type. 
HINT: Do a literature search.  Read primary literature.  Search BIONET
archives (at www.bio.net).  Then ask bionet newsgroups.

2) Choose your transfedction method.  This will also depend on the
cell type.  You may have to try several methods to find one that works
well.  Not everyone can make the same methods work on the same cell
lines.  HINT: Most companies' tech support resources have protocols
for what worked on particular cell lines.

3) Make your construct.  Make sure you're using a vector meant for
stable transfection.  And double check your subcloning strategy (i.e.,
reading frame, orientation, etc.).

4) Purify your DNA.  Not necessarily CsCl pure, but it may have to be.

5) Perform a kill curve.  Use the appropriate antibiotic, and grow the
cells in a number of different concentrations.  They should die after
a couple of passages (a few weeks to a few months, depending on cell
type).

PROTOCOL:

1) Optimize your transfection.  Try a lot of different conditions
(e.g., [DNA], amount of reagent, confluency, etc.).  The more time
spent here, the better.

2) Transfect.  Use your parameters in step 1.

3) Select.  Not immediately!  Select 24-48 hours after transfection. 
This will take months to years.  You are not guaranteed anything at
this step.  You may have to start with a hundred or so clones to get a
few good ones at the end.

4) Maintain.  You will always need to have a small level of antibiotic
present.

Good luck!

rich

==

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Richard J. Dudley
Senior Research Assistant
Center for Genomic Sciences
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