Thermo Scientific KingFisher Plant DNA Kit User manual

Thermo Scientific KingFisher
Plant DNA Kit
Instruction Manual
Rev. 1.1

Thermo Scientific KingFisher
Plant DNA Kit
Instruction Manual
Rev. 1.1, Cat. no. N11996

Copyright © 2011 ermo Fisher Scientic Inc. First edition published in 2010. All rights reserved.
Reproduction of the accompanying user documentation in whole or in part is prohibited.
“BindIt”, “KingFisher”, “Microtiter”, “Multidrop”, “NanoDrop” and "Versette" are registered
trademarks of ermo Fisher Scientic.
All other trademarks and registered trademarks are the property of their respective holders.
ermo Fisher Scientic reserves the right to change its products and services at any time to
incorporate technological developments. is manual is subject to change without prior notice as part
of a continuous product development. Although this manual has been prepared with every precaution
to ensure accuracy, ermo Fisher Scientic assumes no liability for any errors or omissions, nor
for any damages resulting from the application or use of this information. is instruction manual
supersedes all previous editions.
e Product will operate substantially in conformance with ermo Fisher Scientic’s published
specications.
THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES, WHETHER
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business interruption or any consequential, incidental, special or other indirect damages of any kind,
regardless of how caused and regardless of whether an action in contract, tort, strict product liability
orotherwise.

Thermo Fisher Scientific 5Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Contents
Chapter 1 Kit Content ...........................................................................................7
Chapter 2 Product Description.........................................................................11
Introduction ............................................................................11
Intended use .........................................................................11
Principle and procedure ........................................................11
Kit specications ......................................................................12
KingFisher magnetic particle processors ...................................13
Chapter 3 Safety Information............................................................................17
Chapter 4 Storage Conditions and Preparation of
the Working Solution ......................................................................19
Storage conditions ...................................................................19
Preparation of the RNaseA working solution ..........................19
Chapter 5 Protocols and Pipetting Instructions ...........................................21
Handling of KingFisher Magnetic Beads ..................................21
Sample material and storage .....................................................21
Homogenization of sample material .........................................22
Instructions for KingFisher Flex with 96 deep well plates
for DNA purication from 400µl of plant lysate .....................23
Summary of platecontents ...................................................25
Instructions for KingFisher Duo with 12-pin magnet head
and 96 deep well plates for DNA purication from
400µl of plant lysate ...............................................................26
Summary of plate and elution strip contents .........................28
Quantication and determination of the purity of DNA .........29
Chapter 6 General Information.........................................................................31
Reagent specicity and volumes ...............................................31
Handling of magnetic beads ....................................................31
Binding and wash steps ............................................................31
Elution step .............................................................................32

Thermo Fisher Scientific
Contents
6Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Appendix A Troubleshooting ................................................................................35
Appendix B Ordering Information .......................................................................37

Thermo Fisher Scientific 7Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Chapter 1
Kit Content
Table 1-1. Thermo Scientific KingFisher Plant DNA Kit
Item KingFisher Plant DNA Kit
Cat. No. 97050196
Package size 1 x 96 samples
KingFisher Magnetic Beads 3.2 ml
Lysis Buffer 55 ml
Binding Buffer 50 ml
Wash Buffer 1 65 ml
Wash Buffer 2 65 ml
Wash Buffer 3 65 ml
Elution Buffer 20 ml
RNase A (lyophilized) 1 vial
Recommended instrument KingFisher Flex with
96 deep well head
KingFisher Duo
KingFisher 96
e KingFisher Plant DNA Kit (Cat. No. 97050196)
is intended for the purication of 96 samples using the
ermo Scientic KingFisher Flex with a 96 deep well
head or the ermo Scientic KingFisher Duo and
a lysed sample volume of 400µl.

Thermo Fisher Scientific8Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Kit Content
Equipment and reagents to be supplied by the user:
• 80% ethanol (EtOH), molecular biology grade
• DNase-free water
• Homogenization equipment
• Magnetic particle processor
Table 1-2. Thermo Scientific KingFisher magnetic particle processors
Cat. No. Product
5400000 KingFisher magnetic particle processor
5400050 KingFisher mL magnetic particle processor
5400100 KingFisher Duo magnetic particle processor
5400630 KingFisher Flex magnetic particle processor with 96 deep well head
5400640 KingFisher Flex magnetic particle processor with 24 deep well head
Discontinued KingFisher 96 magnetic particle processor
Table 1-3. Thermo Scientific KingFisher Flex consumables
Cat. No. Product Package size
97002514 KingFisher Flex 96 tip comb for PCR magnet 80 pcs
97002524 KingFisher Flex 96 tip comb for KF magnet 100 pcs
97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs
97002610 KingFisher Flex 24 deep well tip comb and plate 50 pcs
97002540 KingFisher Flex 96 KF plate (200 µl) 48 pcs
95040450 Microtiter deep well 96 plate, non sterile 50 pcs
95040460 Microtiter deep well 96 plate, sterile 50 pcs
95040470 KingFisher Flex 24 deep well plate 50 pcs
95040480 KingFisher Flex 24 deep well plate, sterile 50 pcs

Thermo Fisher Scientific 9Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Kit Content
Table 1-4. Thermo Scientific KingFisher Duo consumables
Cat. No. Product Package size
97003500 KingFisher Duo 12-tip comb for Microtiter deep well 96 plate 50 pcs
97003510 KingFisher Duo 6-tip combs and KingFisher 24 deep well plate
(12 pcs of 24 deep well plates, each including 4 tips combs)
48 pcs
97003520 KingFisher Duo elution strip 40 pcs
97003530 KingFisher Duo Combi pack for Microtiter deep well 96 plate
(tips combs, plates and elution strips for 96 samples)
1 box
Table 1-5. Thermo Scientific KingFisher mL consumables
Cat. No. Product Package size
97002111 KingFisher mL tip comb 800 pcs
97002121 KingFisher mL tube 20 x 45 pcs
97002131 KingFisher mL combi (tubes and tip combs for 60 samples) 60
97002141 KingFisher mL combi (tubes and tip combs for 240 samples) 240
Table 1-6. Thermo Scientific KingFisher consumables
Cat. No. Product Package size
97002070 KingFisher tip comb 50 pcs
97002080 KingFisher plate 100 µl 50 pcs
97002084 KingFisher plate 200 µl 50 pcs
97002090 KingFisher plastics 100 µl 8-pack, 8 plates and 8 tip combs 1 box
97002094 KingFisher plastics 200 µl 8-pack, 8 plates and 8 tip combs 1 box

Thermo Fisher Scientific10 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Kit Content

Thermo Fisher Scientific 11Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Chapter 2
Product Description
e KingFisher Plant DNA Kit is designed for rapid
automated purication of DNA from plant materials
using KingFisher instruments. e DNA puried with the
KingFisher Plant DNA Kit is of high quality and free of
proteins, nucleases, and other contaminants or inhibitors.
It is therefore suitable for direct use in many dierent
downstream applications, such as PCR (polymerase
chain reaction), restriction analysis and in several other
enzymatic reactions.
e KingFisher Plant DNA Kit is developed for
the purication of DNA from plant material using
paramagnetic particles. e purication process requires
no phenol/chloroform extraction or alcohol precipitation
and needs very little hands-on time. e reagents and
specic plastic consumables are designed to work with
the KingFisher Flex or KingFisher Duo magnetic particle
processors as part of an integrated system. e KingFisher
Plant DNA Kit is only intended for research use, not
for clinical or diagnostic use. e user is responsible for
validating the performance of the KingFisher instrument
and the KingFisher Plant DNA Kit for any particular use,
because the performance of the kits has not been validated
for any specic organism.
e KingFisher Plant DNA Kit uses magnetic-particle
technology for DNA purication. e ermo Scientic
KingFisher technology combines the speed and eciency
of DNA purication with easy handling of magnetic
particles. It is recommended that fresh, frozen, dried
Introduction
Intended use
Principle and
procedure

Thermo Fisher Scientific12 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Product Description
Kit specifications
or lyophilized sample is mechanically disrupted into a
ne powder before the purication can start to ensure a
good yield of puried DNA. e powder is resuspended
into the Lysis Buer, mixed and incubated and then
sedimented after a short centrifugation step. e cleared
lysates are transferred to ermo Scientic KingFisher
plates for processing with a ermo Scientic KingFisher
magnetic particle processor. DNA binds to the surface
of the ermo Scientic KingFisher Magnetic Beads in
the presence of a chaotropic salt. e following eective
wash steps dispose of proteins, cell debris and any residual
contaminants, while the DNA bound to the KingFisher
Magnetic Beads is transferred through the wash steps.
First, two dierent Wash Buers are used in sequential
wash steps, followed by an ethanol wash. A rapid rinse in
the third Wash Buer removes the residual ethanol and
considerably improves the purity of the DNA. High-
quality DNA is eluted into the Elution Buer and is ready
for subsequent downstream processes, such as enzymatic
reactions. Heat incubation is included in the elution step
when using the KingFisher Flex or KingFisher Duo.
e KingFisher Plant DNA Kit is designed for rapid
automated preparation of highly pure DNA from plant
materials using KingFisher magnetic particle processors.
e approximate processing time is less than 30 minutes
for the purication of 96 samples in the KingFisher Flex
and the KingFisher Duo. e obtained DNA can be
directly used in various downstream applications.
Plant samples can be fresh, frozen, lyophilized or dried.
Asuitable amount of fresh material is 20–50 mg. For
dried material, the amount of sample should be one fth
of the amount of fresh sample. e yields of acquired
puried DNA depend on the sample type, the method of
sample collection, and the method of tissue disruption.
e KingFisher Plant DNA Kit can be processed at
room temperature excluding the homogenization with
Kit specifications

Thermo Fisher Scientific 13Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Product Description
KingFisher magnetic particle processors
Lysis Buer and Proteinase K treatment, which occurs
outside the KingFisher instrument and requires heating.
Additionally, heating during the elution step may increase
the DNA yield by approximately 15–20%.
e KingFisher Magnetic Beads are highly reactive,
superparamagnetic beads. e binding capacity is 0.4µg
of DNA per 1µl of bead suspension.
e KingFisher magnetic particle processors are designed
for the automated transfer and processing of magnetic
particles in microplate format. e patented technology
of the ermo Scientic KingFisher systems is based
on the use of magnetic rods covered with a disposable,
specially designed tip comb and plates. Use only ermo
Scientic KingFisher plastic consumables. Use of
products from other manufacturers may cause unsuitable
mixing or even instability in the KingFisher instrument.
e instrument functions without any dispensing
or aspiration parts or devices. Samples and reagents,
including magnetic particles, are dispensed into the plates
according to the corresponding instructions. Dispensing
can be done manually or partially automatically using
automatic dispensers, for example, the ermo Scientic
Multidrop Combi and/or the ermo Scientic Versette.
ermo Scientic BindIt Software 3.2 can be used for
running ready-made and optimized protocols for the
ermo Scientic KingFisher Kits. It is also possible to
transfer the dened protocol onto the onboard software
and run it directly from the instrument. e KingFisher
instruments oer a rapid and automated solution for
complicated and time-consuming purication processes
without risk of carryover or cross contamination,
resulting in high-purity DNA.
e KingFisher instrument family comprises four systems
covering working volumes from 20 to 5000µl. Each
system consists of an instrument, specially designed plastic
consumables and the easy-to-use BindIt Software 3.2.
KingFisher magnetic
particle processors

Thermo Fisher Scientific14 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Product Description
KingFisher magnetic particle processors
eKingFisher Plant DNA Kit is optimized and ready for
use with KingFisher systems.
e KingFisher magnetic particle processors are intended
for professional research use by trained personnel. Detailed
information and user instructions for the KingFisher
instruments can be found in their respective user manuals.
Table 2-1. Overview of Thermo Scientific KingFisher systems
KingFisher Flex
96 format 24 format
KingFisher Duo
12 format 6 format
Processing volume 20–1000µl 200–5000µl 50–1000 µl 200–5000 µl
Capacity Up to 96 samples per
run (lysed sample
volume 400µl with
the KingFisher
PlantDNA Kit)
Up to 24 samples
per run
Up to 12 samples
per run
Up to 6 samples per
run (sample volume
3 ml)
Magnet heads 96 interchangeable
formats for
PCR plate,
KingFisherFlex96
KF plate, Microtiter
deep well 96 plate
24 format for
KingFisher 24 deep
well plate
12-pin magnet head
for Microtiter deep
well 96 plate
6-pin magnet head
for KingFisher Flex
24 deep well plate
Plates KingFisher Flex96
KF plate(20–200µl),
Microtiter deep well
96 plate(50–1000µl),
96 well PCR plate,
skirted(20–100µl)
KingFisher Flex 24
deep well plate
(200–5000µl)
Microtiter deep well
96 plate (50–1000µl),
KingFisher Duo
elution strip
(30–130µl)
KingFisher Flex 24
deep well plate
(200–5000µl)
Tip combs KingFisher Flex96
tip comb for
PCR magnets,
KingFisherFlex
tip comb for
KFmagnets,
KingFisher Flex96
tip comb for deep
well magnets
KingFisher Flex 24
tip comb for deep
well magnets
KingFisher Duo
12-tip comb
KingFisher Duo
6-tip comb
Heating
temperature
Heating block temperature from +5°C
above ambient room temperature
to +115°C
Heating block temperature from +10°C
to +75°C, elution strip +4°C to +75°C in
room temperature

Thermo Fisher Scientific 15Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Product Description
KingFisher magnetic particle processors
Table 2-1. Overview of Thermo Scientific KingFisher systems
KingFisher mL KingFisher
Processing volume 50–1000µl 20–200µl
Capacity Up to 15 samples per run Up to 24 samples per run
Magnet heads 15 format 24 format
Plates KingFisher mL tube, special tube strip with
1 x 5 tubes (50–1000µl)
KingFisher plate 100or 200µl (20–100µl
or 20–200µl)
Tip combs KingFisher mL tip comb 1 x 5 format KingFisher tip comb 1 x 12 format
Heating
temperature
No heating available No heating available
e BindIt Software 3.2 protocol optimized for the
KingFisher Plant DNA Kit is available for the KingFisher
Flex or KingFisher Duo. BindIt Software 3.2 protocols
for the ermo Scientic KingFisher 96, the KingFisher
and the ermo Scientic KingFisher mL are available
on request. For more information, contact your local
authorized distributor.

Thermo Fisher Scientific16 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Product Description
KingFisher magnetic particle processors

Thermo Fisher Scientific 17Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Chapter 3
Safety Information
e following components of the KingFisher Plant DNA
Kit contain hazardous contents (Table 3-1).
Wear a laboratory coat, disposable gloves and goggles, and
follow the safety instructions given in the kit instruction
manual. It is recommended that Good Laboratory Practice
(GLP) is followed to guarantee reliable analyses.
Table 3-1. Safety precautions
Reagent Hazardous contents Safety instructions
Binding Buffer Isopropanol > 90% Highly flammable. Irritating to eyes. Vapors may
cause drowsiness and dizziness. Keep container
tightly closed. Keep away from sources of
ignition–no smoking. Avoid contact with skin and
eyes. In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice.
Wash Buffer 1 Sodium perchlorate< 15%
and ethanol<24%
Flammable.
Wash Buffer 2 Sodium perchlorate< 15%
and ethanol < 24%
Flammable.
RNase A Lyophilized RNase A May cause sensitization by inhalation and skin
contact. Keep container tightly closed.
Do not inhale dust.

Thermo Fisher Scientific18 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Safety Information

Thermo Fisher Scientific 19Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Chapter 4
Storage Conditions and
Preparation of the Working
Solution
All buers and reagents included in the KingFisher Plant
DNA Kit can be stored at room temperature (20–25°C)
and are stable for up to one year from the manufacturing
date. e buers are ready for use.
To prepare the RNase A enzyme for the ermo Scientic
KingFisher Plant DNA Kit (Cat. No. 97050196), add
1.25ml of DNase-free water to the RNase A vial. Store at
+4°C in aliquots.
Storage conditions
Preparation of
the RNaseA
working solution

Thermo Fisher Scientific20 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Storage Conditions and Preparation of the Working Solution
Preparation of the RNaseA working solution