User guide

Methods
Creating expression plasmids for the Freedom
™
DG44 Kit
Introduction
The Freedom
™
DG44 Kit contains two vectors, pOptiVEC
™
-TOPO
®
and
pcDNA
™
3.3-TOPO

. See pages 66–69 for maps and features of each vector.
Using the instructions in this manual, you will:
• For two subunits proteins, clone two separate PCR products corresponding to
each of the two subunits (SU1 and SU2, SU: subunit) of your protein of choice
separately into pOptiVEC
™
-TOPO
®
TA and pcDNA
™
3.3-TOPO

TA vectors to
create four expression plasmids. Since individual protein expression may
depend on the combination of vectors containing the different subunits of
your protein of interest, you will optimize these conditions using different
combinations of clones.
• For single subunit proteins, create an expression plasmid using the
pOptiVEC
™
-TOPO
®
TA.
For more information on creating expression plasmids, refer to the instructions in
the pOptiVEC
™
-TOPO
®
TA and pcDNA
™
3.3-TOPO

TA Cloning Kit manuals
(part nos. 25-0977 and 25-1010, respectively), which are available at
www.lifetechnologies.com/manuals.
Types of expression
plasmids
You will create two expression plasmids in each vector for a total of four
expression plasmids for two subunit proteins, or create one expression plasmid
for a single subunit protein:
Vector DNA Selection
pcDNA
™
3.3-TOPO

TA mammalian secretion signal and SU1
Geneticin
®
reagent
pcDNA
™
3.3-TOPO

TA mammalian secretion signal and SU2
Geneticin
®
reagent
pOptiVEC
™
-TOPO
®
TA mammalian secretion signal and SU1 DHFR/HT-
pOptiVEC
™
-TOPO
®
TA mammalian secretion signal and SU2 DHFR/HT-
Recommendation
The combination of vectors for transfection into mammalian cells and the
selection process for the Freedom
™
DG44 Kit are described on the following
pages. For an overview of the experimental steps required to express your protein
of interest and the various common experimental pathways you may take while
using the Freedom
™
DG44 Kit, see Experimental flowcharts on pages 8–9.
14