Optimization of the DNA Purification
Protocol for the Thermo Scientific
KingFisher Flex and BindIt 3.1 Software
SP & A Application laboratory, Thermo Fisher Scientific, Vantaa, Finland
Overview
Purpose: Optimal quantity and quality of DNA with
the magnetic particle based extraction technology
Method: Thermo Scientific KingFisher Flex magnetic
particle processor and BindIt® 3.1 Software
Results: By simple optimization of the nucleic acid
extraction protocol it is possible to achieve a great
yield of high quality DNA
Introduction
Rapid and efficient isolation of nucleic acids
from complex biological matrixes is an important
step to get optimal starting material for various
experiments. KingFisher®, the magnetic bead based
automated purification system, provides a quick
and easy solution to achieve reproducible results in
purification of pure and intact DNA or RNA with
minimal hands-on time. The KingFisher technology
is based on magnetic rods transferring particles
through the various purification phases – binding,
mixing, washing and elution.
The KingFisher is an open and flexible system; the
user can choose any available magnetic particle
based purification kit suitable for the application.
The purification protocol is created with the BindIt
3.1 Software enabling the user to optimize the
purification conditions by taking into consideration
the sample type and particles used in the extraction
kit. The latest member of the KingFisher family is
the KingFisher Flex that offers both high throughput
and a wide range of processing volumes.
It is important to optimize the purification protocol
to reach the optimal quantity and quality of nucleic
acids with the magnetic particle based technology.
All the purification steps require beads to separate
effectively, and the mixing speeds are significant for
optimal binding, efficient washing and active elution.
In this technical note we present some guidelines
for generating an ideal DNA purification protocol
for KingFisher Flex. Different mixing combinations
and the effect of heating in the elution were tested,
and the quantity and quality of purifications were
compared. Wide range of the processing volumes
with the KingFisher Flex are possible because several
different magnetic heads and plate formats are available. In this study we have used 96-deep well
and 24-well magnet heads and tip combs. The DNA
quantity was measured by reading the absorbance
at 260 nm and the quality by analyzing the 260/280
nm ratio. The quality of DNA was also tested by
an end-point PCR to control the presence of PCR
inhibitors in the eluate.
The data shows that the optimization of the
KingFisher Flex purification protocol enhances
the quality and quantity of the extracted DNA.
The protocol is easy to modify with the BindIt 3.1
Software, which is available for all the KingFisher
instrument models.
Material and methods
A KingFisher Flex DNA purification protocol
typically consists of cell lysis / DNA binding,
several washing steps and DNA elution. All these
purification steps were optimized for both the 96-
and 24-deep well format. In the 96-well format
the DNA extraction was performed from 200 μl of
blood or ˜6 μg of pure calf thymus DNA, and in the
24-well format from 1 mL of blood or ˜35 μg of
calf thymus DNA. The kits used in the experiments
were InviMag® Blood DNA kit (Invitek, Germany)
and BioSprint DNA Blood kit (Qiagen, Germany).
The absorbance of the eluates was measured with
the Thermo Scientific NanoDrop 8000 and the DNA
yield was calculated based on the measured 260 nm
absorbance.
The protocols were optimized by changing one
variable at a time. The mixing speed of one step was
optimized by alternating the available mixing speeds
while the rest of the protocol remained unchangeable.
BindIt 3.1 Software (Fig. 2) was used for making
modifications to the isolation protocols.
Results
Faster mixing speeds increase DNA yields for the
DNA purification from blood. When binding,
washing and elution were tested one variable at
a time, the faster mixing speeds for all the steps
resulted in better yield (Table 1). The mixing
speed causes a bigger difference to the results with
the KingFisher Flex 24-well format, because of
the shaping of the tip and the well. The protocol
parameters in the binding and elution steps affected
primarily the total DNA yield. In the washing steps
the SLOW mixing had an effect on the DNA quality.
The purified DNA was analyzed with the PCR and the results show the remaining impurities affecting
the secondary applications (Fig. 3). Table 1 shows
the effects of different mixing speeds on the bind and
elution steps and the effect of the heating and elution
volume on the final DNA yield.
Conclusions
The different mixing speeds have a strong effect
on the DNA purification with the KingFisher Flex
magnetic particle processor. For the DNA purification
it is important to mix the solutions efficiently to
reach optimal binding between the DNA and the
bead. Powerful washing is essential for the high purity
of the DNA. Results of the SLOW mixing speed
indicate impurities in the elution and inhibition of
the enzymatic reaction due to inefficient washes. The
slower mixing speeds are frequently preferred during
the elution due to the DNA degradation, but according
to our experiments the FAST mixing in the elution results in the best quantity and quality of DNA. The
SLOW mixing speed is useful for the heated steps of
the nucleic acid purification.
These test results correspond to the DNA
purification. However, the same optimizing is
not directly applicable to e.g. RNA and protein
purification on the KingFisher Flex. HALF MIX,
MEDIUM or SLOW mixing speeds are recommended
for more sensitive bead-biomolecule complexes.
Summary
- KingFisher Flex provides a rapid and reliable
method for DNA isolation
- Easy adjustment of isolation protocols with BindIt
3.1 Software
- Open platform is simple to adapt for all available
magnetic particle separation kits