1. science
2. biology
3. molecular biology

Smarter Nucleic Acid Sample Preparation
on Thermo Scientific KingFisher Systems
Content
InviMag® Technology……………………………………………………………….
Isolation Principles………………………………………………………………….
Product Selection Guide for KingFisher mL kits………………………………....
Product Selection Guide for KingFisher 96 and KFflex kits…………………….
3
4
5
7
01// Cancer Genetics
DNA isolation from blood and body fluids………………………………………...
DNA sample preparation from stool samples…………………………………….
9
9
02// Genotyping
DNA isolation from tissue samples and cells……………………………………..
DNA isolation from a variety of plant species…………………………………….
GMO analysis……………………………………………………………………….
10
10
11
03// Pathogen Detection
Nucleic acid purification from viruses……………………………………………...
Nucleic acid purification from bacteria and viruses………………………………
Bacterial DNA from different sample types………………………………………..
DNA sample preparation from saliva and swabs…………………………………
DNA sample preparation from stool samples……………………………………..
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04// Gene Expression Analysis
RNA isolation from different starting materials…………………………………...
16
05// Cloning Experiments
Efficient plasmid DNA isolation…………………………………………………….
17
References…………………………………………………………………………..
Ordering Information………………………………………………………………..
18
19
1
Smarter Nucleic Acid Sample Preparation
STRATEC Molecular offers a broad range of products for magnetic bead based applications, which are
developed for highly efficient purification of DNA and RNA on the KingFisher® workstations from Thermo
Scientific.
Long years of experience in kit development and reagent optimization in combination with a thorough
understanding of requirements and limitations in automation have resulted in highly optimized solutions
for your automated nucleic acid extraction needs.
General purification procedure
First the samples are lysed in an optimized lysis buffer according to kit instructions. The lysis can be
performed on the KingFisher 96 or Flex platform which allows for the sample incubation due to the
integrated heating block in the instrument. In the case of KingFisher mL system, the lysis is performed
outside of the instrument using external heating blocks. All subsequent steps after lysis, such as binding
of the nucleic acid on the InviMag® beads, washing and final DNA/RNA elution are automatically
performed on the KingFisher platform in a fast and reproducible manner.
InviMag® kits provide all necessary reagents and plastic ware for sample preparation. Furthermore the
kits can be ordered without the KingFisher plastics (see ordering information pages 19/20).
Advantages
•
•
•
•
•
purification of a large variety of samples from multiple sources
fast & cross contamination free process
high throughput - up to 96 samples per run
ready to use and customized purification protocols
large surface of magnetic beads, highly reactive magnetic particles with high binding capacity
resulting in exceptional recovery and yield
Implementation
•
•
protocols are simply downloaded from www.invitek.de
special program solutions can be tailored for customers on request
 Contact us: +49 30 9489 2901/2910 or [email protected]
 or your local sales representative
2
InviMag® Technology
The InviMag® technology combines the advantages of innovative nucleic acid purification chemistries
(non-chaotropic, RTP® and PSP®) with the easy handling of magnetic particles for a highly efficient and
reliable purification of high purity nucleic acids.
3
Isolation Principles
4
Product Selection Guide for KingFisher mL kits
Nucleic acid
genomic
DNA
Starting material
1 - 200 µl fresh, frozen or old human
or other mammalian whole blood
(EDTA, citrate)
1 – 25 µl non-mammalian blood
up to 30 µl buffy coat
up to 20 µl bone marrow
up to 200 µl rinse liquid from swabs
Product name
File name
®
InviMagBloodDNAKFmL_KF.
msz
®
InviMag SalivaGene DNA
KFmL_KF.msz
InviMag Blood DNA Mini Kit/ KFmL*
®
500 µl SalivaGene DNA Stabilizer /
Saliva Mix 1:1, 500 µl Swab
®
SalivaGene DNA Stabilizer
100 mg (homogenized) fresh, frozen
or dried plant material
InviMag SalivaGene DNA Kit/ KFmL
®
InviMag Plant DNA Mini Kit/ KFmL
InviMag Plant DNA
KfmL_KF.msz
9
bacterial
DNA
10 bacterial cells,
1-10 mg tissue, biopsy sample, FFPE
tissue
1-100 μl blood,
15 - 50 ml urine,
200 μl cell-free body fluids,
1 l water
®
InviMag Bacteria DNA Mini Kit/ KFmL
200 mg fresh or frozen stool sample or InviMag® Stool DNA Mini Kit/ KFmL*
~1 g sample in 8 ml Stool Stabilizer
viral
DNA/RNA
200 μl serum, plasma, cell-free body
fluids, cell culture supernatant
InviMagStoolDNAKFmL_KF.
msz
®
InviMagVirusDNARNAKFmL_KF.msz
®
InviMagVirusDNAKFmL_KF.
msz
®
InviMagVirusRNAKFmL_KF.
msz
®
on request
InviMag Virus DNA/RNA Mini Kit/ KFmL*
400 µl rinse liquid from swabs
1-10 mg tissue
up to 200 μl serum or plasma,
whole blood, cell-free body fluids,
rinse liquid from swabs,
cell culture supernatant
InviMag Virus DNA Kit/ KFmL*
viral RNA
up to 200 µl serum or plasma,
cell-free body fluids, rinse liquid from
swabs
up to 50 mg stool samples
up to 10 mg tissue
InviMag Virus RNA Kit/ KFmL*
total RNA
up to 5 x 10 cells
up to 1.0 ml blood
up to 20 mg tissue sample
viral DNA
InviMagBacteriaKFmL_KF.m
sz
6
InviMag Universal RNA Mini Kit/ KFmL
5
20 - 25 min
after lysis
50 μg, depending on starting material
A260:A280 1.8 – 2.0
20 - 25 min
after lysis
depending on viral titer
limit of sensitivity: ~500 DNA/RNA virus
copies/ml
depending on viral titer
limit of sensitivity: ~500 DNA virus copies/ml
depending on viral titer
limit of sensitivity: ~500 RNA virus copies/ml
20 μg, depending on starting material
A260:A280 1.6 – 2.0
20 - 25 min
after lysis
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Northern
Blot
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RT-PCR
RFLPAnalysis
depending on starting material
A260:A280 1.8 – 2.0
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20 - 25 min
after lysis
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SNPAnalysis
5 - 25 µg, depending on type of starting
material
A260:A280 1.8 – 2.0
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STRAnalysis
20 - 25 min
after lysis
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HLA-typing
10 µg, depending on starting material
A260:A280 1.8 – 2.0
Southern
Blot
20 - 25 min
after lysis
RE
Digestion
up to 10 µg (in average about 6 µg),
depending on amount of lymphocytes,
sample source, sample transport, sample
storage, and age of the sample
A260:A280 1.8 – 2.0
Sequencing
Processing time
PCR/
Real-time
PCR
Yield and ratio
20 - 25 min
after lysis
20 - 25 min
after lysis
30 min after lysis
*) developed and manufactured in compliance with the Directive 98/79/EC on in vitro diagnostic medical devices (IVD-Directive). Products
which are CE-marked according to the IVD-Directive can be used for diagnostic applications in countries where this directive is recognized.
6
Product Selection Guide for KingFisher 96 and KFflex kits
Nucleic acid
Starting material
Product name
®
plasmid
DNA
1 ml (mini)-10 ml (midi) bacterial
suspension; bacterial pellets from 1 ml
(mini) – 10 ml (midi) of bacterial
suspension
up to 200 μl (mini) - 1 ml (midi) whole
human blood (EDTA, citrate)
up to 25 µl (mini) – 125 µl (midi) non
mammalian blood, up to 30 µl (mini) –
150 µl (midi) buffy coat, up to 20 µl
bone marrow (mini)
genomic
DNA
5 - 20 mg tissue sample, rodent tail,
FFPE tissue,
6
10-10 mammalian cells
InviMag Plasmid Mini Kit/ KF96
®
InviMag Plasmid Midi Kit/ KFflex 24
®
InviMag Blood DNA Mini Kit/ KF96*
®
InviMag Blood DNA Midi Kit/ KFflex 24*
®
InviMag Tissue DNA Mini Kit/ KF96*
®
500 µl SalivaGene DNA Stabilizer /
Saliva Mix 1:1, 500 µl Swab
®
SalivaGene DNA Stabilizer
100 mg (homogenized) fresh, frozen or
dried plant material
®
InviMag SalivaGene DNA Kit/ KF96
®
InviMag Plant DNA Mini Kit/ KF96
9
bacterial
DNA
bacterial
DNA, viral
DNA/RNA
viral
DNA/RNA
viral DNA
InviMag Blood DNA Midi Kit
Flex24_KF
InviMag Tissue DNA
KF96_KF
InviMag Tissue DNA
KFflex96_KF
InviMag_Salivagene_Human
_KF96.kf2
InviMag_Salivagene_Bacteria
_KF96.kf2
InviMag Salivagene DNA
Human KFflex96_KF.msz
InviMag Salivagene DNA
Bacteria KFflex96_KF.msz
InviMagPlantDNAKF96_KF.m
sz
InviMagPlantDNAKFflex96_K
F.msz
InviMagUniBacteriaKF96_KF.
msz
InviMagUniBacteriaKFflex96_
KF.msz
200 mg fresh or frozen stool sample or
~1 g sample in 8 ml Stool Stabilizer
InviMagStoolDNAKF96_KF.m
sz
InviMagStoolDNAKFflex96_K
F.msz
200 μl serum, plasma, cell-free body
fluids, cell culture supernatant, rinse
liquid from swabs, up to 10 mg tissue,
9
up to 10 bacterial cells
200 μl serum, plasma, cell-free body
fluids, cell culture supernatant, up to
50 µl blood , up to 10 mg tissue, up to
50 µl grinded plant material
200 μl (mini)-1 ml (midi) serum, plasma,
whole blood, cell-free body fluids, rinse
liquid from swabs
®
InviMag Stool DNA Kit/ KF96*
®
InviMag Pathogen Kit/ KF96
®
InviMag Virus DNA/RNA Mini Kit/ KF96
®
InviMag Virus DNA Kit/ KF96*
®
InviMag Virus DNA Midi Kit/ KFflex 24
InviMag Virus RNA Kit/ KF96*
200 μl (mini) -1 ml (midi) serum,
plasma, cell-free body fluids, rinse liquid
from swabs Mini kit: 10 mg tissue
®
InviMag Virus RNA Midi Kit/ KFflex 24
sample, 50 mg stool sample
6
total RNA
InviMagPlasmidDNAKF96_K
F.msz I
InviMagPlasmidDNAKFflex96
_KF.msz
InviMag Plasmid DNA
KFflex24_KF.msz
InviMagBloodDNAKF96_KF.
msz
InviMagBloodDNAKFflex96_
KF.msz
10 bacterial cells, 1-10 mg tissue,
biopsy sample, FFPE tissue, 1-100 μl
®
blood, 200 μl cell-free body fluids, 15-50 InviMag Universal Bacteria Kit/ KF96
ml urine, 1 l water
®
viral RNA
File names
up to 5 x 10 cells
up to 1.5 ml blood
up to 10 - 40 mg tissue sample
®
InviMag Universal RNA Mini Kit/ KF96
7
InviMag Pathogen Kit/ KF96
InviMag Pathogen Kit/
KFflex96
InviMagVirusDNARNAKF96_KF.msz
InviMagVirusDNARNAKFflex96_KF.msz
InviMagVirusDNAKF96_KF.m
sz
InviMagVirusDNAKFflex96_K
F.msz
InviMag Virus DNA Midi Kit
KFflex24_KF.msz
InviMagVirusRNAKF96_KF.m
sz
InviMagVirusRNAKFflex96_K
F.msz
InviMag Virus RNA
KFflex24_KF.msz
InviMag Universal RNA
KF96.kf2
InviMag Universal RNA
KFflex96_KF.msz
about 50 min
50 μg, depending on starting material
A260:A280 1.8 – 2.0
about 50 min
depending on viral titer and starting material
limit of sensitivity: ~500 DNA/RNA virus
copies/ml
depending on viral titer
limit of sensitivity: ~500 DNA/RNA virus
copies/ml
about 30 min after
lysis
depending on viral titer
limit of sensitivity: ~500 DNA virus copies/ml
20 - 25 min
50 min after lysis
depending on viral titer
limit of sensitivity: ~500 RNA virus copies/m
20 - 25 min
40 min after lysis
up to 80 µg, depending on starting material
A260:A280 1.7 – 2.1
about 60 min
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RFLPAnalysis
depending on starting material
A260:A280: 1.7 – 2.0
■
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20 - 25 min
after lysis
■
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5 - 25 μg depending on type of starting
material
A260:A280 1.8 – 2.0
■
■
40 – 50 min
■
SNPAnalysis
10 μg, depending on starting material
A260:A280 1.7 – 2.0
■
STRAnalysis
20 - 25 min
after lysis
■
HLA-typing
10 - 25 μg, depending on type, storage and
source of tissue sample
A260:A280 1.8 – 2.0
Southern
Blot
20 - 25 min
after lysis
■
RE
Digestion
3 - 12 μg (mini); 15 - 20 μg (midi), depending
on protocol, age, storage and source of blood
sample
A260:A280 1.8 – 2.0
■
Northern
Blot
20 - 25 min
30 - 35 min
after lysis
RT-PCR
5 - 10 µg (mini); 70 - 100 µg (midi) per ml
overnight culture, depending on plasmid and
host strain
A260:A280 1.7 – 2.0
Sequencing
Processing time
PCR/
Real-time
PCR
Yield and ratio
*) developed and manufactured in compliance with the Directive
98/79/EC on in vitro diagnostic medical devices (IVD-Directive).
Products which are CE-marked according to the IVD-Directive can
be used for diagnostic applications in countries where this directive
is recognized.
01//
Cancer Genetics
DNA isolation from blood and body fluids
The InviMag® Blood DNA Mini Kits are designed for genomic DNA purification from human and animal
blood as well as from buffy coat, bone marrow or rinse liquid from swabs. Fresh or frozen whole blood
treated with EDTA or citrate, from routine blood collection systems can be used.
patient
control
patient
control
A
Fig. 1
B
Detection of Dihydropyrimidin Dehydrogenase (DPYD) Exon 14 skipping mutations by PCR
®
®
DNA was isolated from 150 µl EDTA blood on the KingFisher mL using the InviMag Blood DNA Mini Kit/ KFmL. 2 µl of the
®
eluted DNA (1/50) were used in a qualitative PCR (figure A) on a LightCycler . Figure B shows the first derivation of the melting
curve of the amplified product. If the melting curve shows two maxima a DPD mutation is detected.
Red line:
Green Line:
patient sample
heterocygote control
(Data kindly provided by H. Hannig, Central Institution for Molecular Diagnostics, Clinic Braunschweig gGmbH, Germany)
DNA sample preparation from stool samples
The InviMag® Stool DNA Kits are designed for DNA isolation from pathogenic microorganisms and from
the host organism from up to 200 mg of stool sample or about 1 g sample in 8 ml Stool DNA Stabilizer.
In this solution the DNA samples are stable at room temperature for 3 days for easy transport and
storage. DNases are inactivated and the microorganism titer is preserved.
A
Fig. 2
B
Detection of mutations in the K-ras Gene, based on non-invasive patient samples by sequence analysis
on an ABI 3170
®
DNA from 50 mg of fecal samples from 12 different humans was isolated using the InviMag Stool DNA Kit/ KF96. The total
DNA recovery [ng/µl] was measured by Nanodrop and the amount of human DNA was detected by real-time PCR [pg/ul]. The
average amount for the total DNA of the 12 samples was 159.1 ng/prep, including 0.114 % genomic DNA. 5 µl aliquot were
analyzed for integrity on an agarose gel (figure A). For the analysis of point mutation in the K-ras gene 5 µl aliquot can be used
in standard K-ras-PCR. Figure B shows a sequence analysis of point mutation in the K-ras gene using the isolated DNA in a
specific PCR including the relevant part of the K-ras sequence. The upper sequence analysis shows at codon 12 the wild type
with a very small part of mutated sequence (C T exchange).
9
02//
Genotyping
DNA isolation from tissue samples and cells
The InviMag® Tissue DNA Mini Kit/ KF96 enables genomic DNA purification from a variety of tissue
types incl. mouse and rat tails, cryosections, food and from eucaryotic cells. PCR inhibitors, which can
interfere with the amplification reaction, are efficiently removed. Pure DNA can be used in subsequent
downstream applications, such as PCR, real-time PCR, SNP detection, sequencing and cloning.
Fig. 3
Ma
1a
2a
3a
Mb 1b 2b
DNA isolation from mouse tail
3b
®
DNA was isolated using the InviMag Tissue DNA Mini
Kit/ KF96 from 0.4 cm mouse tail (different diameters).
The samples were lysed on a thermomixer for 1.5 h at
56 °C and the DNA was eluted in 200 µl Elution Buffer.
5 µl of the eluate were amplified in a 25 µl PCR reaction.
10 kb
3 kb
2 kb
Lane Ma – GeneRuler™ DNA Ladder, Fermentas
Lane 1a - 10 µl of genomic DNA
Lane 2a - 10 µl of genomic DNA
Lane 3a - 10 µl of genomic DNA
500 kb
Lane Mb - GeneRuler™ DNA Ladder, Fermentas
Lane 1b - 5 µl of the PCR product
Lane 2b - 5 µl of the PCR product
Lane 3b - 5 µl of the PCR product
02//
Plant Genotyping
DNA isolation from a variety of plant species
The InviMag® Plant DNA Mini Kits are an effective solution for isolation of high-quality total cellular DNA
from a wide variety of plant species and tissue types (e.g. leaves, roots, fruits, flowers, wood, seeds,
water plants, GMO, food). Furthermore the kit is suitable for co-purification of DNA from
phytopathogens. Up to 100 mg of fresh or frozen material can be processed in one hour. The isolated,
pure DNA can be used in subsequent downstream applications such as PCR, restriction analysis, realtime PCR, cloning, genotyping etc.
1
2
3
4
5
6
7
8
Lane 1, 8
Lane 2 - 4
Lane 4 - 7
Fig. 4
GeneRuler™ DNA Ladder, Fermentas
DNA from Allium cepaand
DNA from Hedera helix
Analysis of plant DNA
DNA from 60 mg of Allium cepaand and from 60 mg Hedera helix was isolated using the InviMag Plant DNA Mini Kit/
KFmL on the KingFisher mL system. 10 µl of the eluted DNA were analysed, after an RNase A treatment, on a 0.8 % agarose
gel, stained with ethidium bromide.
10
02//
Plant Genotyping - GMO Analysis
Genomic DNA from corn
Positive control
Standard
Sample
Red curve – positive control
Light green curves – samples
Dark green curves – standard
GMO determination
Positive control
Sample
Standard
Red curve – positive control
Light blue curves – samples
Dark blue curves – standard
Fig. 5
Identification of GMOs in corn compounds
®
DNA was isolated from 60 mg hackled corn compounds including defined amounts of GMO material, using the InviMag Plant
DNA Mini Kit/ KFmL. The DNA was eluted in 100 µl Elution Buffer. 5 µl of the eluted DNA were used for the real-time
amplification of genomic DNA and GMO-DNA, to determine the copy number. By measurement of genomic and GMO copies
the relation between the two genetic components was calculated. It was compared with the known data of the materials.
Percentage of GMO copies to DNA copies
Corn
compounds
A
Genomic DNA copies
GMO DNA
copies
0
Ratio
Genomic DNA: GMO DNA
0
Theoretical amount of
GMO copies
0
B
C
15.009
5
0.03 %
0.02 %
11.556
23
0.2 %
0.3 %
D
9.422
70
0.74 %
1.0 %
E
10.205
527
5.2 %
8.0 %
10.715
(Data kindly provided by S. Mergemeier, Congen Biotechnologie GmbH, Germany)
11
03//
Pathogen Detection
Nucleic acid purification from viruses
The InviMag® Virus DNA/RNA Mini Kits are designed for reliable, simultaneous isolation of high quality
viral DNA and RNA from a variety of starting materials using a lyophilized, solid lysis buffer incl.
Proteinase K and Carrier RNA (stable at room temperature!). The use of this buffer reduces handling
steps, infectious plastic waste and processing time. The Carrier RNA enhances nucleic acid recovery in
samples with low viral titer, allowing for sensitive virus detection in qualitative and quantitative assays.
For purification of only one nucleic acid type the InviMag® Virus RNA Kits and the InviMag® Virus DNA
Kits are also available (both kits use a common liquid lysis buffer).
Mean Ct
value
Std.
deviation
31.52
0.16
32.47
0.12
InviMag Virus DNA/RNA Mini
Kit/ KF96
32.67
0.13
RTP® DNA/RNA Virus Mini Kit
32.48
0.28
Positive control
32.23
0.36
Sample
InviMag® Virus RNA Kit/ KF96
®
InviMag Virus DNA/RNA Mini
Kit/ KFmL
®
Fig. 6
Comparison of different extraction methods for viral RNA purification
®
Viral RNA was isolated using the InviMag Virus DNA/RNA Mini Kits on KingFisher mL and KingFisher 96 in comparison to the
®
®
spin format (RTP DNA/RNA Virus Mini Kit) and the InviMag Virus RNA Kit on the KingFisher 96. Human plasma samples
were spiked with same lot of Influenza A virus with a dilution of 1:200. 200 µl starting material were used for the viral RNA
extraction. 2.5 µl of the eluted RNA were analyzed in triplicates using Influenza A-specific real-time PCR. The Virus RNA kit
shows higher sensitivity but the Virus DNA/RNA kit can be used for both DNA and RNA viral purification.
Fig. 7
Isolation and detection of BVD-Virus from plasma pools
®
RNA from bovine diarrhea virus from cattle was isolated from 5 ml pooled plasma sample (5 ml) using the InviMag Virus
DNA/RNA Kit/ KFmL on the KingFisher mL system. Only 100 µl of plasma sample per animal of maximum 50 animals were
used to produce the 5 ml pools. From each pool an aliquot of only 200 µl serum or plasma were used for viral RNA isolation.
Routinely a 5 µl aliquot of each eluate (1/24) was finally amplified in a subsequent real-time PCR to detect BVDV infection from
the pools, down to 10 genomic copies per reaction and is in the range of sensitivities required by the German government. This
corresponds approximately to a dilution of a persistently infected animal (PI) sample of at least 1:1000.
(Data kindly provided by H. Nieper; LUA Leipzig, Germany)
12
03//
Pathogen Detection
Nucleic acid purification from viruses
Positive control
Norovirus infected patient sample
Negative control
Fig. 8
Norovirus RNA isolation from human stool samples
®
Viral RNA was isolated from 50 mg of Norovirus infected fecal sample using the InviMag DNA/RNA Virus Mini Kit /KFmL.
®
10 µl of the eluted RNA were analyzed in a specific real-time RT-PCR LightCycler .
(Data kindly provided by H. Hannig, Central Institution for Molecular Diagnostics, Clinic Braunschweig gGmbH, Germany)
Nucleic acid purification from bacteria and viruses
The InviMag® Pathogen Kit/ KF96 provides an universal nucleic acid purification system for pathogens one kit for bacterial DNA and viral DNA/RNA purification allowing simultaneous testing of both virus
types and bacteria. This kit uses a lyophilized lysis buffer (incl. Proteinase K, Lysozyme, Carrier RNA)
which is stable at room temperature. The use of this buffer reduces handling steps, infectious plastic
waste and processing time. The Carrier RNA enhances nucleic acid recovery in samples with low viral
titer, allowing for sensitive virus detection in qualitative and quantitative assays.
A
Fig. 9
B
Simultaneous isolation of viral RNA and DNA
A plasma pool was spiked with a defined titer of Influenza A (1 µl ++ plasma / 200 µl) and HBV (500 copies / 200 µl) and viral
®
DNA and RNA were isolated simultaneously from 200 µl aliquots using the InviMag Pathogen Kit/ KF96 on a KingFisher Flex
96. 5 µl of the eluates were used in virus specific RT-PCR (A - detection of Influenza A) and real-time PCR (B – detection of
HBV) reactions.
(Blue - virus samples; red - positive controls)
13
03//
Pathogen Detection
Bacterial DNA from different sample types
The InviMag® Bacteria Kits are designed for genomic DNA isolation from different bacteria species from
a broad range of starting materials. Pure DNA can be used in subsequent downstream applications,
such as PCR, real-time PCR, restriction analysis, and sequencing. The InviMag® Bacteria DNA Mini Kit/
KFmL for use on KingFisher mL contains the pre-filled Extraction Tubes with lyophilized lysis
components (lysis buffer, Lysozyme, Proteinase K) and Carrier RNA; stable at room temperature. After
lysis the samples are transferred to the magnetic bead based procedure with a PCR template
preparation time of 25 min.
patient sample
controls
Fig. 10
patient samples
Detection of Mycobacteria tuberculosis infections from human sputum samples
Isolation of bacterial DNA from 100 µl of a two week old bacterial culture, which was infected by patient samples (clinical
®
samples: sputum, bronchial lavage) using the InviMag Bacteria DNA Mini Kit/ KFmL. 5 µl of the eluted DNA were used for the
detection of Mycobacteria tuberculosis complex or directly of Mycobacteria tuberculosis by real-time application (16s - RNA
fragment) on a LightCycler.
(Data kindly provided by H. Hannig, Central Institution for Molecular Diagnostics, Clinic Braunschweig gGmbH, Germany)
10
10
10
Fig. 11
3
4
5
Detection of Salmonella
BPW-media was inoculated with 50 cells of Salmonella typhimurium and cultivated. A defined dilution series was prepared from
®
1 ml overnight culture and DNA was isolated using the InviMag Bacteria DNA Mini Kit/ KFmL on the KingFisher mL system.
5 µl of the eluted DNA were used for the detection of Salmonella typhimurim by real-time PCR.
(Data kindly provided by I. Wiemann, Congen Biotechnologie GmbH, Germany)
14
03//
Pathogen Detection
DNA sample preparation from saliva and swabs
The SalivaGene® system combines the use of the novel SalivaGene® Collector or the SalivaGene®
Buccal Swab for saliva and swab sample collection and subsequent DNA purification. The SalivaGene®
Collector and the SalivaGene® Buccal Swab tubes are pre-filled with a DNA stabilization buffer which
eliminates the need of sample cooling and stabilizes genomic DNA for 12 months at room temperature.
The non-invasive saliva and swab collection methods reduce puncture-associated infection risks and
provide high quality DNA performing as well as blood-derived DNA in downstream applications. The
SalivaGene® system further realizes the prelysis of bacteria and a very efficient and fast isolation of
high-quality total DNA.
Sample
Fig. 12
mean Ct value
Std. deviation
KFflex 96
22.59
0.67
KFmL
20.50
0.42
Positive control
25.09
0.34
®
Bacterial DNA isolation from SalivaGene stabilized saliva samples
5
500 µl of different saliva samples were spiked with 5 µl of Bacillus subtilis overnight culture (approx. 5 x 10 cells per sample)
®
and stabilized using the SalivaGene DNA stabilization buffer. The samples were stored at RT for three months. The DNA was
®
isolated using the InviMag SalivaGene DNA Kit on the KFmL and KFflex 96 using the standard protocols and analyzed using
a specific real-time PCR.
DNA sample preparation from stool samples
The InviMag® Stool DNA Kits are designed for DNA isolation from pathogenic microorganisms and from
the host organism from up to 200 mg of stool sample or about 1 g sample in 8 ml Stool DNA Stabilizer.
In this solution the DNA samples are stable at room temperature for 3 days for easy transport and
storage. DNases are inactivated and the microorganism titer is preserved.
K+306 positive control
K0:
negative control
1 - 4:
template-DNA, isolated from fecal samples from rats
5 - 8:
template-DNA, isolated from fecal samples from gerbilis
L:
Marker, 100 bp ladder (Invitrogen)
(Data kindly provided by Ms. Lorenz, GIM GmbH, Wildenbruch,
Germany)
Fig. 13
Health Monitoring - detection of Helicobacter bilis from rats and gerbils
®
DNA was isolated from 150-200 mg feces from rats and gerbiles using the InviMag Stool DNA Kit/ KF96 on the KingFisher 96
system. The isolated DNA was used for PCR detection of Helicobacter ssp, in this special experiment for the detection of
Helicobacter bilis (Fragment sizes: 638 bp) and analyzed on a 1.5 % agarose gel. (Fox et al., J Clin. Microbiol. (1995) 33, 445454: Helicobacter bilis sp. nov., a Novel Helicobacter Species Isolated from Bile, Livers, and Intestines of Aged, Inbred Mice).
The lanes without PCR product show the animals without any H. bilis infection.
15
04//
Gene Expression Analysis
RNA isolation from different starting materials
For gene expression analysis high-quality RNA is essential for downstream assays, such as RT-PCR
and microarray analysis. The InviMag® Universal RNA Mini Kits for automated RNA purification are
suitable for use with a wide range of cell types, tissue and blood samples. The genomic DNA is
removed without an enzymatic digestion step. RNases are inactivated to prevent RNA degradation. The
kits provide a highly efficient and cost-effective total RNA purification system. Yields of total RNA are
highly reproducible and comparable to common spin systems.
B
Figure A
Figure B
Fig. 14
Reproducible isolation of high-quality RNA
For RNA isolation bovine oviduct cells after first passage were cultured to confluence in 6 well plates and exposed to media
with different nutrient compositions for 12 h, 24 h or 48 h, respectively. Highly pure total RNA was isolated using the InviMag
Universal RNA Mini Kit on the KingFisher Flex 96. The preparation time for 96 samples in parallel takes approx. 1 h –
1 h 30 min. A representative result for the RNA purity and integrity (RIN) is shown (A). An aliquot of each eluate was used for a
real-time RT-PCR reaction for GAP-DH. All samples showed amplification signals and the expression pattern of the housekeeping gene is shown in (B).
1) The high purity of the isolated RNA is shown in a RIN Factor of about 10.0.
2) The yield of total RNA was between 10 - 20 µg per well depending on the animal and growing rate.
3) The ratio A260: A280 is in average 2.05 (standard deviation +/-0.2%).
(Data kindly provided by C. Gabler and C. Holder, Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany)
16
05//
Cloning Experiments
Efficient plasmid DNA isolation
The InviMag® Plasmid DNA procedure for isolation of low and high copy plamids combines an easy-toautomate magnetic bead technology with a novel plasmid purification method. The unique chemistry
used in the InviMag® procedure enables purification of plasmid DNA directly from crude lysate without
the need for a lysate clearing step (centrifugation), allowing automation of the entire purification
procedure. Under the optimized buffer conditions, plasmid DNA selectively binds to the surface of the
magnetic beads, while genomic DNA and cellular debris stay in the solution.
pUCpol
pGEM
pUCpol
pGEM
pUCpol
KF96
KF Flex96
pGEM
KF96
pUCpol pGEM
KF Flex96
A
Fig. 15
B
Convenient pDNA isolation using magnetic beads without centrifugation steps
Isolation of plasmid DNA from bacterial pellet from 1 ml overnight culture of E.coli DH5a pUCpol, E.coli XL1 pGEM and E.coli
®
XL1 without plasmid using the InviMag Plasmid Mini Kit/ KF96 on the KingFisher 96 and Flex systems. 10 µl of the eluates
were analyzed on a 0.8 % agarose gel (Figure A). Figure B shows an example restriction enzyme digest with Hind III of one of
each plasmid shown in A and analysis on a 0.8 % agarose gel. Shown are the isolated pDNA and the respective Hind III digest
for the same sample.
Plasmid DNA isolation from large sample volumes
The InviMag® Plasmid Midi Kit/ KFflex 24 is designed for semi-automated preparation of plasmid DNA
from up to 10 ml overnight culture using magnetic beads and the KingFisher Flex24 workstation. Up to
24 samples in parallel can be processed.
1
2
3
4
5
6
7
Lane 1
Lane 2,3
Lane 4,5
Lane 6,7
10 kb ladder
isolated pDNA from 1 ml overnight culture
isolated pDNA from 5 ml overnight culture
isolated pDNA from 10 ml overnight culture
Fig. 16
Plasmid DNA clones (E.coli strain XL-1, vector pGEM, 3 kb plasmid) were purified from bacterial overnight culture
®
using the InviMag Plasmid Midi Kit/ KFflex 24 on the KingFisher Flex24 system. 10 µl of the eluate were analyzed on a 1 %
agarose gel.
17
References
®
InviMag Blood DNA Mini Kit/ KFmL
Chantratita et al.; Journal of Clinical Microbiology, 2008; 46 (2): 568-573. Loop-Mediated Isothermal Amplification Method Targeting the TTS1
Gene Cluster for Detection of Burkholderia pseudomallei
Chantratita et al.; Am. J. Trop. Med. Hyg., 2007; 77 (5): 814-817. Prospective Clinical Evaluation of the Accuracy of 16S rRNA Real-Time PCR
Assay for the Diagnosis of Melioidosis
Hannig et al.; Thermo Scientific Application Note Lit. No.: AP-MIB-KFML01-0907, September 2007. Analysis of the Mutation in the Human
DPYP Gene from Human Blood Using the InviMag Blood Mini Kit with the Thermo Scientific KingFisher mL Instrument
Lamberg et al.; Thermo Scientific Technical Note Lit. No.: TN-MIB-FRS01-0506. Purification of Forensic DNA Samples with the KingFisher
Magnetic Particle Processor and Characterization of the Purified DNA
®
InviMag Blood DNA Mini Kit/ KF96
Lamberg et al.; Thermo Scientific Technical Note Lit. No: TN-MIB-FRS01-0506. Purification of Forensic DNA Samples with the KingFisher
Magnetic Particle Processor and Characterization of the Purified DNA
®
InviMag Blood DNA Midi Kit/ KFflex 24
Suomalainen et al.; Thermo Scientific Application Note Lit. No.:AP-MIB-KFFLEX-0508 Rapid and reproducible DNA Isolation from 1 ml of
whole blood with Thermo Scientific KingFisher Flex
®
InviMag Plant DNA Mini Kit/ KFmL
Liefting et al.; Bulletin of Insectology 64 (Supplement): S93-S94, 2011, ISSN 1721-8861. An update on phytoplasma diseases in New Zealand
Liefting et al.; Australasian Plant Pathol.DOI 10.1007/s13313-011-0036-z. New hosts of “Candidatus Phytoplasma australiense” in New
Zealand
Liefting et al.; Plant Disease 2010 94:4, 479-479. First Report of Helleborus net necrosis virus in Hellebore in New Zealand
Ward et al.; MAF Biosecurity New Zealand Technical Paper No: 2010/14 Prepared for MAFBNZ Operational, ISBN 978-0-478-37045-4 (print),
ISBN 978-0-478-37046-1 (online), ISSN 1179-5832 (print), ISSN 1179-5840 (online), July 2010. Development of a LAMP assay for Xylella
fastidiosa
Liefting et al.; Plant Disease 2009 93:9, 969-969. First Report of ‘Candidatus Phytoplasma australiense’ in Potato
InviMag Stool DNA Mini Kit/ KFmL
Nylund et al.; J Microbiol Methods. 2010 Nov;83(2):231-5. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic
microarray analysis
®
InviMag Bacteria DNA Mini Kit/ KFmL
Kozak et al.; J Bacteriol. 2010 Feb;192(4):1030-44. Virulence factors encoded by Legionella longbeachae identified on the basis of the genome
sequence analysis of clinical isolate D-4968
Hannig et al.; Thermo Scientific Application Note Lit. No.: AP-MIB-KFML01-0907, September 2007. High-sensitive detection of Mycobacterium
tuberculosis from human clinical samples using the InviMag Bacteria DNA Mini Kit/ KFmL with the Thermo Scientific KingFisher mL instrument
®
InviMag Virus DNA/RNA Mini Kit/ KFmL
Nieper et al.; Thermo Scientific Application Note Lit. No.: AP-MIB-KF01-0407, April 2007. High-Sensitive Isolation of the BVD Virus from Bovine
Plasma Pools
18
Ordering Information
Product name
®
InviMag Plasmid Mini Kit/ KF96
®
InviMag Plasmid Midi Kit/ KFflex 24
®
InviMag Blood DNA Mini Kit/ KFmL
®
InviMag Blood DNA Mini Kit/ KF96
®
InviMag Blood DNA Midi Kit/ KFflex 24
®
InviMag Tissue DNA Mini Kit/ KF96
®
InviMag SalivaGene DNA Kit/ KFmL
®
InviMag SalivaGene DNA Kit/ KF96
®
InviMag Plant DNA Mini Kit/ KFmL
®
InviMag Plant DNA Mini Kit/ KF96
®
InviMag Bacteria DNA Mini Kit/ KFmL
®
InviMag Universal Bacteria Kit/ KF96
®
InviMag Stool DNA Mini Kit/ KFmL
®
InviMag Stool DNA Kit/ KF96
®
InviMag Virus DNA/RNA Mini Kit/ KFmL
®
InviMag Virus DNA/RNA Mini Kit/ KF96
®
InviMag Virus DNA Kit/ KFmL
®
InviMag Virus DNA Kit/ KF96
®
InviMag Virus DNA Midi Kit/ KFflex 24
®
InviMag Virus RNA Kit/ KFmL
®
InviMag Virus RNA Kit/ KF96
®
InviMag Virus RNA Midi Kit/ KFflex 24
®
InviMag Universal RNA Mini Kit/ KFmL
®
InviMag Universal RNA Mini Kit/ KF96
Catalogue number
7410300100
7410300200
7410710100
7410710200
2431110100
2431110200
2431110300
2431110400
7431300100
7431300200
7431710100
7431710200
7432300100
7432300200
2435160100
2435160200
7435060100
7435060200
2437110100
2437110200
2437110300
2437110400
7437300100
7437300200
2433150100
2433150200
7433300100
7433300200
2438110100
2438110200
7438300100
7438300200
2441150100
2441150200
2441150300
2441150400
7441050100
7441050200
2442110100
2442110200
7412300100
7412300200
7442710100
7442710200
2443110100
2443110200
2443110300
2443110400
7443300100
7443300200
7443710100
7443710200
2460110100
2460110200
7460300100
7460300200
19
Size
1 x 96 purifications
5 x 96 purifications
1 x 24 purifications
5 x 24 purifications
15 purifications
75 purifications
150 purifications
300 purifications
1 x 96 purifications
5 x 96 purifications
1 x 24 purifications
5 x 24 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
150 purifications
300 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
150 purifications
300 purifications
1 x 96 purifications
5 x 96 purifications
15 purifications
75 purifications
1 x 96 purifications
5 x 96 purifications
1 x 24 purifications
5 x 24 purifications
15 purifications
75 purifications
150 purifications
300 purifications
1 x 96 purifications
5 x 96 purifications
1 x 24 purifications
5 x 24 purifications
15 purifications
75 purifications
1 x 96 purifications
5 x 96 purifications
Ordering Information – without KingFisher plastics
Product name
®
InviMag Plasmid Mini Kit/ KF96 wp
®
InviMag Plasmid Midi Kit/ KFflex 24 wp
®
InviMag Blood DNA Mini Kit/ KFmL wp
®
InviMag Blood DNA Mini Kit/ KF96 wp
®
InviMag Blood DNA Midi Kit/ KFflex 24 wp
®
InviMag Tissue DNA Mini Kit/ KF96 wp
®
InviMag SalivaGene DNA Kit/ KFmL wp
®
InviMag SalivaGene DNA Kit/ KF96 wp
®
InviMag Plant DNA Mini Kit/ KFmL wp
®
InviMag Plant DNA Mini Kit/ KF96 wp
®
InviMag Bacteria DNA Mini Kit/ KFmL wp
®
InviMag Universal Bacteria Kit/ KF96 wp
®
InviMag Stool DNA Mini Kit/ KFmL wp
®
InviMag Stool DNA Kit/ KF96 wp
®
InviMag Virus DNA/RNA Mini Kit/ KFmL wp
®
InviMag Virus DNA/RNA Mini Kit/ KF96 wp
®
InviMag Virus DNA Kit/ KFmL wp
®
InviMag Virus DNA Kit/ KF96 wp
®
InviMag Virus DNA Midi Kit/ KFflex 24 wp
®
InviMag Virus RNA Kit/ KFmL wp
®
InviMag Virus RNA Kit/ KF96 wp
®
InviMag Virus RNA Midi Kit/ KFflex 24 wp
®
InviMag Universal RNA Mini Kit/ KFmL wp
®
InviMag Universal RNA Mini Kit/ KF96 wp
Catalogue number
7410300250
7410300350
7410710250
7410710350
2431110250
2431110350
2431110450
7431300250
7431300350
7431710250
7431710350
7432300250
7432300350
2435160250
2435160350
2435160450
7435060250
7435060350
2437110250
2437110350
2437110450
7437300250
7437300350
2433150250
2433150350
2433150450
7433300250
7433300350
2438110250
2438110350
2438110450
7438300250
7438300350
2441150250
2441150350
2441150450
7441050250
7441050350
2442110250
2442110350
2442110450
7442300250
7442300350
7442710250
7442710350
2443110250
2443110350
2443110450
7443300250
7443300350
7443710250
7443710350
2460110250
2460110350
2460110450
7460300250
7460300350
20
Size
5 x 96 purifications
10 x 96 purifications
5 x 24 purifications
10 x 24 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
5 x 24 purifications
10 x 24 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
5 x 24 purifications
10 x 24 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
5 x 24 purifications
10 x 24 purifications
75 purifications
150 purifications
300 purifications
5 x 96 purifications
10 x 96 purifications
STRATEC Molecular GmbH
STRATEC Molecular develops and manufactures reagents and kits for DNA/RNA stabilization,
extraction and purification using manual and automated systems. Established under the name Invitek in
1992, STRATEC Molecular provides innovative system solutions for nucleic acid sample preparation
from any sample type. The company is internationally respected for its outstanding and high
performance technology platforms and offers a broad spectrum of DNA/RNA sample preparation kits
and reagents for molecular diagnostics and life science research.
Together with STRATEC Biomedical's proven and reliable instrumentation experience STRATEC
Molecular will develop automated system solutions for tomorrow´s challenges in molecular diagnostic
sample preparation.
STRATEC Molecular has implemented a quality system certified according to EN ISO 13485:2003 +
AC:2009 and ISO 9001:2008. In addition, STRATEC Molecular products are subject to extensive quality
controls. A large number of STRATEC Molecular products are CE-marked*) in compliance with the
Directive 98/79/EC on in vitro diagnostic medical devices (IVD-Directive). STRATEC Molecular
guarantees that all products will perform as indicated and will provide the highest quality of support.
About the STRATEC Group
The STRATEC group consists of the publicly listed parent company STRATEC Biomedical AG and of
subsidiaries and second-tier subsidiaries in Germany, the USA, the UK, Switzerland and Romania. The
STRATEC Biomedical AG (http://www.stratec.com) designs and manufactures fully automated systems
for its partners in the fields of clinical diagnostics and biotechnology.
*) Products which are CE-marked according to the IVD-Directive can be used for diagnostic applications in countries where this directive is
recognized.
21
STRATEC Molecular GmbH
Robert-Rössle-Str. 10
13125 Berlin, Germany
Phone: +49 30 9489 2901/ 2910
Fax: +49 30 9489 2909
E-mail: [email protected]
www.invitek.de
®
®
®
InviMag , RTP , PSP and SalivaGene® are
registered trademarks of STRATEC Molecular.
Other brands or product names are
trademarks of their respective holders.
© 2012 STRATEC Molecular, all rights reserved.
6G7o06/KingFisher/06/2012
YOUR LOCAL DISTRIBUTOR:
22
STRATEC Molecular GmbH
Robert-Rössle-Str. 10
D-13125 Berlin, Germany
Phone: +49 30 9489 2901
Fax: +49-30-9489 2909
E-mail: [email protected]
www.invitek.de