Biomedical Engineering Reference
In-Depth Information
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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
j
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 microtiter 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
j
Avoid excess exposure to light since the dye is light sensitive.
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Alternatively, the DNA concentration can be calculated from measuring the A
260 nm
using
a spectrophotometer (e.g. Nanodrop). However, for quantification of DNA from FFPE tissue
the use of Picogreen gives more reliable estimates than measurement of A
260 nm
using a
spectrophotometer because only double-stranded DNA (and no degradation products) are
measured.
PROTOCOL 3.4 DNA Quality Control PCR
Equipment and reagents
Primer stocks (100
μ
M
):
— RS2032018 (150 bp)
•
primer 1: 5
-GTGTCTCCCTTCCCACTCAA-3
◦
primer 2: 5
-AGCCCACCTACCTTGGAAAG-3
◦
