9. Application Note for DNA Quantification
A commonly used DNA probe,
PicoGreen
TM
allows direct quantitation of PCR
amplicons without purification from the reaction mixture and makes it possible to
detect low levels of DNA contamination in recombinant protein products. In
comparison to the Hoechst 33258 dye, which shows significant AT selectivity, the
PicoGreen reagent shows little if any AT- or GC-selectivity and is thus accurate
for quantitating DNA from almost any source.
We demonstrate here an example for the quantitation of DNA, comparing
absorbance (260 nm) with fluorescence detection using
PicoGreen
TM
. We are
able to show the advantage to have instrumentation available for both detection
techniques, dependent on the yield of extracted DNA. The DNA samples used for
the measurements were plasmid DNA and genomic DNA, extracted with the
TECAN
GENESIS
RSP 150.
9.2 Experimental
Setup
9.2.1 DNA
extraction
•
Plasmid extraction
Plasmid extraction from
E.coli
(DH5
α
) with a bluescript vector with a 1.2 kb insert
grown in LB or TB media was performed using the QIAprepR 96 Turbo BioRobot
Kit from QIAGENR and the QIA_96 Turbo T Gemini script on a
GENESIS
RSP 150
with a
Te-VacS-B
from TECAN.
•
Genomic DNA Extraction
Genomic DNA was extracted from human blood samples using a Genomic DNA
Extraction Kit on a
GENESIS
RSP 150 with a
Te-MagS
from TECAN.
9.2.2 Assay
Procedure
The extracted DNA was quantified using UV absorbance at 260 nm and
fluorescence measurements with PicoGreen
TM
(from Molecular Probes Inc.) at
485/535 nm with the TECAN GENios. Absorbance measurements were
performed in special UV compatible plates (GREINER UV Star® Nr. 655801),
and for fluorescence quantitation, black flat bottom 96 well plates (GREINER; Nr
655076) were used.
For
UV absorbance at 260 nm
, 100
µ
l of DNA solution per well was used. In
addition to the samples, a
λ
DNA dilution series was pipetted onto the same plate
to calculate a calibration curve. The
fluorescence measurements with
PicoGreen
TM
dye were performed refering to the protocol for the PicoGreen
dsDNA Quantiation Kit (Molecular Probes,
). The solutions were mixed
by shaking for 5 sec prior to the measurement. A dilution series of
λ
DNA was
used as calibration standard.
9-2
Operating Manual for GENios, GENios FL, and GENios
Plus
No: I 112 904 Rev No: 1.1
June 2002
