Biology Reference
In-Depth Information
15. Plate 1-2 × 10
5
cells/well in 24-well plates or1×10
6
cells/well in 6-well plates.
The volume of the culture medium in each well should be kept low. Volumes
of 0.5 and 2 mL are sufficient for individual wells of 24- and 6-well plates,
respectively.
16. At this point, the cell lysate can be stored at -20 °C for months before continuing
on with the rest of the protocol.
17. The
dotA/
pCR2.1-TOPO plasmid is 6349 bp in size (2418 bp
dotA
PCR
product plus 3931 pCR2.1-TOPO vector). One microgram of this plasmid equals
0.24 pmoles which equals 1.44 × 10
11
plasmid molecules (i.e.,
dotA
copies).
Determine the concentration of a stock solution of plasmid spectrophotometri-
cally. Make a dilution series of the plasmid such that a 10 μL aliquot of each
diluted stock results in a range of 10
3
-10
8
plasmid copies.
Acknowledgments
We thank Dale Howe and Diane Cockrell for critical review of this manuscript.
This research was supported by the Intramural Research Program of the National
Institutes of Health, National Institute of Allergy and Infectious Diseases.
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