Biomedical Engineering Reference
In-Depth Information
PROTOCOL 1.3 DNA Concentration Measurement Using Picogreen
Equipment and reagents
TE: 10 m
M
Tris-HCl, pH 8.0, 0.1m
M
EDTA
•
PicoGreen dsDNA reagent(Molecular Probes)
d
•
Lambda DNA standards
•
Recommended microtiter plates (immunoassay microplates - flat bottom; Dynex
Immulux
TM
)
•
Fluorescence plate reader
•
Centrifuge for microtiter plates.
•
e
Method
1 Prepare a series of 100
μ
l/well lambda DNA standards, in duplicate, within a clean,
96-well plate as follows:
Lamba DNA (2
μ
g/
μ
l)
TE (
μ
l)
Final concentration
100
μ
l
0
100 ng/
μ
l
75
μ
l
25
750 pg/
μ
l
50
μ
l
50
500 pg/
μ
l
25
μ
l
75
250 pg/
μ
l
10
μ
l
90
100 pg/
μ
l
5
μ
l
95
50 pg/
μ
l
0
μ
l
100
0 pg/
μ
l
2 For each DNA sample, prepare duplicate dilutions of 2
μ
lofDNAwith98
μ
lofTE.
3 For each DNA sample dilution, prepare 100
μ
l of Picogreen
d
reagent by diluting
Picogreen 200-fold in TE.
4 Add 100
μ
l of diluted Picogreen reagent to each diluted DNA sample and mix by
pipetting up and down.
5 Centrifuge the microtitre plate at 250
g
for 1 min to remove possible bubbles.
6 Read in a plate reader (excitation 485 nm, emission 538 nm).
7 Calculate concentrations from the standard curve using the plate reader software
package.
Notes
d
Avoid excess exposure to light since the dye is light sensitive.
e
For quantitation of DNA from FFPE tissue for SNP arrays, the use of Picogreen gives more
reliable estimates than measurement of A
260 nm
using a spectrophotometer [12].
