Thermo Scientific KingFisher Plant DNA Kit Instruction 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
EXPRESSED OR IMPLIED, ORAL OR WRITTEN, WITH RESPECT TO THE PRODUCTS,
INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF PRODUCT
QUALITY, CONDITION, DESCRIPTION, MERCHANTABILITY OR FITNESS FOR ANY
PARTICULAR PURPOSE. THERMO FISHER SCIENTIFIC DOES NOT WARRANT THAT
THE PRODUCTS ARE ERROR-FREE OR WILL ACCOMPLISH ANY PARTICULAR RESULT.
THERMO FISHER SCIENTIFIC HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY
REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS,
INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE, INVALID OR
INCOMPLETE RESULTS.

ermo Fisher Scientic and its aliates shall have no liability to an End User arising out of the
use or inability to use the product, including, without limitation, for any loss of use or prots,
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

6 Thermo 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 Scientific8 Thermo 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. High­quality 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

Thermo Fisher Scientific 21Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Chapter 5

Protocols and Pipetting
Instructions

Before beginning the DNA purication protocol,
carefully read through the ermo Scientic KingFisher

Flex User Manual (Cat. No. N07669), the ermo
Scientic KingFisher Duo User Manual (Cat. No. N12420)
and the ermo Scientic BindIt Software for KingFisher
Instruments version 3.2 UserManual (Cat. No. N07974).

BindIt Software 3.2 protocols for the KingFisher, the
KingFisher mL and the KingFisher 96 can be obtained on
request.

A homogeneous distribution of the KingFisher Magnetic
Beads in the container is essential before the beads are
transferred to the wells or tubes in order to ensure a
high consistency between the wells or tubes. To gain
complete resuspension of the beads, shake the container
vigorously or vortex briey. e KingFisher Magnetic
Beads have a tendency to sediment relatively quickly in
the Binding Buer. Once a premixture of the beads and
the Binding Buer has been made, the mixture should be
used immediately to avoid the risk of transferring variable
amounts of the beads to the respective wells or tubes.

Use 20–50mg of fresh plant sample as sample material.
With dried material, use only one fth of the starting
material compared to the fresh sample material. For
example, instead of using 30mg of fresh plant leaf, use
only 6mg of dried plant leaf.

Handling of

KingFisher

Magnetic Beads

Sample material

and storage

Thermo Fisher Scientific22 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Homogenization of sample material

Using young plant samples, and/or if possible keeping
plants for 12 hours in darkness before collecting the
samples, reduces the polysaccharide and polyphenolic
contents, which may interfere in downstream applications.
Plant samples can be stored in frozen or lyophilized form,
or in ethanol.

Ecient homogenization of the sample material is an
essential step before DNA purication in order to gain
a good yield of high-quality DNA. Plant tissue can be
homogenized, for example, with a pestle, using steel
beads or with commercial homogenizers, of which
high-throughput homogenizers oer a suitable method for
handling 96 samples simultaneously. e homogenization
step must disrupt the structures of the starting material
rapidly and completely in order to ensure a high yield of
DNA.

Homogenize 20–50mg of fresh plant sample (or less if
dried samples are used). After homogenization, add 500µl
of Lysis Buer to a homogenized sample and mix for
30seconds. If RNaseA treatment is needed to reduce the
amount of RNA in the sample, add RNaseA to the Lysis
Buer at a nal concentration of 0.25mg/ml. RNaseA
treatment is recommended for the samples containing
large amounts of RNA. Centrifuge the sample briey in
the Lysis Buer (30seconds, 1500 x g) and incubate at
56°C for 30minutes.

To clear the plant lysate, centrifuge the samples for
20minutes (6000 x g). Transfer the supernatant to a
ermo Scientic Microtiter deep well 96 plate and begin
the purication of DNA using the KingFisher Flex or
KingFisher Duo. Seethe detailed instructions below.

Homogenization of

sample material

Thermo Fisher Scientific 23Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Homogenization of sample material

ese instructions are for the DNA purication from
400µl of plant lysate using the KingFisher Plant DNA Kit
(Cat. No. 97050196, 1 x 96) and the KingFisher Flex with
96 deep well plates.

1. Homogenize the samples according to the instructions

given in "Homogenization of sample material" on

page22.

2. Take ve empty Microtiter deep well 96 plates and two

empty ermo Scientic KingFisher Flex 96 KF plates.

3. Prepare ve Microtiter deep well 96 plates and one

KingFisher Flex 96 KF plate as indicated in the table
below. Resuspend the KingFisher Magnetic Beads well
(e.g., by vortexing) before transferring them from the
bottle.

Plate
number

Plate
type

Plate
name

Content Sample/reagent

volume per well

1 Microtiter

deep well
96 plate

Sample Plant lysate

KingFisher
Magnetic Beads

Binding Buffer

400 µl

30 µl

400 µl

2 Microtiter

deep well
96 plate

Wash 1 Wash Buffer 1 600 µl

3 Microtiter

deep well
96 plate

Wash 2 Wash Buffer 2 600 µl

4 Microtiter

deep well
96 plate

Wash
EtOH

80% ethanol 600 µl

5 Microtiter

deep well
96 plate

Wash 3 Wash Buffer 3 600 µl

6 KingFisher

Flex 96
KFplate

Elution Elution Buffer 150 µl

Instructions for

KingFisher Flex

with 96 deep well

plates for DNA

purification from

400µl of plant lysate

Thermo Fisher Scientific24 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Homogenization of sample material

4. Place a ermo Scientic KingFisher Flex 96 tip comb
for deep well magnets on a Tip Plate (that is, an
empty KingFisher Flex 96 KF plate).

5. Start the KF_PlantDNA_Flex96 protocol with the
KingFisher Flex 96 and load the plates.

Switch on the KingFisher Flex and make sure that you
are using the KingFisher Flex 96 deep well head and
heating block. Connect the PC with BindIt Software 3.2
to the KingFisher Flex. Start the KF_PlantDNA_Flex96
protocol. Insert the Tip Plate and the lled plates into the
instrument as indicated on the KingFisher Flex display.
After all the plates have been loaded into the instrument,
the protocol will begin.

When the KingFisher Flex is to be run as a standalone
instrument, transfer the KF_PlantDNA_Flex96
protocol to the KingFisher Flex. e instructions for
transferring the protocol can be found in Chapter 4:
“Using thesoftware” in the BindIt Software for KingFisher
Instruments version 3.2 User Manual.

6. After the run is completed, remove the plates and store
the puried DNA.

When the protocol is completed, remove the plates
according to the instructions on the KingFisher Flex
display and turn o the instrument. Store the puried
DNA accordingly. e puried DNA is ready for use in
downstream applications.

e nal DNA concentration in the Elution Buer may
increase if the puried DNA is eluted into a smaller
volume of the buer than is recommended, but this can
slightly reduce the overall DNA yield.

Thermo Fisher Scientific 25Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Homogenization of sample material

Table 5-1. Summary of plate contents

Plate
number

Plate
type

Plate
name

Content Sample/reagent

volume per well

1 Microtiter

deep well
96 plate

Sample Plant lysate

KingFisher
Magnetic Beads

Binding Buffer

400 µl

30 µl

400 µl

2 Microtiter

deep well
96 plate

Wash 1 Wash Buffer 1 600 µl

3 Microtiter

deep well
96 plate

Wash 2 Wash Buffer 2 600 µl

4 Microtiter

deep well
96 plate

Wash
EtOH

80% ethanol 600 µl

5 Microtiter

deep well
96 plate

Wash 3 Wash Buffer 3 600 µl

6 KingFisher

Flex 96
KFplate

Elution Elution Buffer 150 µl

7 KingFisher

Flex 96
KFplate

Tip Plate

Summary of

platecontents

Thermo Fisher Scientific26 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Instructions for KingFisher Duo with 12-pin magnet head and 96 deep well plates for DNA purification from 400µl of plant lysate

ese instructions are for the DNA purication from
400µl of plant lysate using the KingFisher Plant DNA
Kit (Cat. No. 97050196) and the KingFisher Duo with a
12-pin magnet head and 96 deep well plates.

1. Homogenize the samples according to the instructions
given in "Homogenization of sample material" on

page22.

2. Take one empty Microtiter deep well 96 plate and one
ermo Scientic KingFisher Duo elution strip.

3. Prepare the Plant DNA plate (Microtiter deep well
96plate).

Add the following reagents to the rows. Note that row B
is reserved for the tip comb and should be left empty.
Note that row C and D are left empty. Resuspend the
KingFisher Magnetic Beads well (e.g., by vortexing) before
transferring them from the bottle.

Plate name and type Row Row name Content Reagent/Sample

volume per well

Plant DNA plate

Microtiter deep well
96 plate

A Sample Plant lysate

KingFisher
Magnetic Beads

Binding Buffer

400 µl

30 µl

400 µl

B Tip 12-tip comb Empty

C Empty Empty Empty

D Empty Empty Empty

E Wash 1 Wash Buffer 1 600 µl

F Wash 2 Wash Buffer 2 600 µl

G Wash_EtOH 80% ethanol 600 µl

H Wash 3 Wash Buffer 3 600 µl

Instructions for
KingFisher Duo

with 12-pin magnet

head and 96 deep

well plates for DNA

purification from

400µl of plant lysate

Thermo Fisher Scientific 27Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Instructions for KingFisher Duo with 12-pin magnet head and 96 deep well plates for DNA purification from 400µl of plant lysate

4. Fill the KingFisher Duo elution strip as follows.
Make sure that the elution strip is placed in the
correct direction into the elution block. Ensure that
the perforated end is facing towards the user and the
Elution Buer is pipetted into the correct wells.

Elution strip Content Reagent volume

per well

KingFisher Duo elution strip Elution Buffer 100 µl

5. Place a ermo Scientic KingFisher Duo 12-tip
comb into row B on a Plant DNA plate.

6. Start the KF_PlantDNA_Duo protocol with the
KingFisher Duo and load the plate and elution strip.

Switch on the KingFisher Duo and make sure that you
are using the KingFisher Duo 12-pin magnet head and
heating block. Connect the PC with BindIt Software 3.2
to the KingFisher Duo. Start the KF_PlantDNA_Duo
protocol. Insert the Plant DNA plate and elution strip
into the instrument as indicated on the KingFisher Duo
display and press OK. Make sure that the elution strip
is placed in the correct direction into the elution block.
Ensure that the perforated end is facing towards the user.

When the KingFisher Duo is to be run as a standalone
instrument, transfer the KF_PlantDNA_Duo protocol
to the KingFisher Duo. e instructions for transferring
the protocol can be found in the BindIt Software for
KingFisher Instruments version 3.2 User Manual.

7. After the run is completed, remove the plate and store
the puried DNA.

When the protocol is completed, remove the plate
and elution strip according to the instructions on the

Thermo Fisher Scientific28 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Instructions for KingFisher Duo with 12-pin magnet head and 96 deep well plates for DNA purification from 400µl of plant lysate

KingFisher Duo display and turn o the instrument.
Store the puried DNA accordingly. e puried DNA is
ready for use in downstream applications. e nal DNA
concentration in the Elution Buer may increase if the
puried nucleic acid is eluted into a smaller volume of the
buer than is recommended, but this can slightly reduce
the overall DNA yield.

Table 5-2. Summary of plate and elution strip contents

Plate name and type Row Row name Content Reagent/Sample

volume per well

Plant DNA plate

Microtiter deep well
96 plate

A Sample Plant lysate

KingFisher
Magnetic Beads

Binding Buffer

400 µl

30 µl

400 µl

B Tip 12-tip comb Empty

C Empty Empty Empty

D Empty Empty Empty

E Wash 1 Wash Buffer 1 600 µl

F Wash 2 Wash Buffer 2 600 µl

G Wash_EtOH 80% ethanol 600 µl

H Wash 3 Wash Buffer 3 600 µl

Elution strip Elution Elution Buffer 100 µl

Summary of plate and

elution strip contents

Thermo Fisher Scientific 29Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Quantification and determination of the purity of DNA

It is recommended to measure the absorbance at 320 nm,
280nm, and 260nm. e concentration of DNA can
be dened with the absorbance at 260nm (A). One
unit at 260nm corresponds to 50µg of DNA per ml.
e ratio between the A/A indicates the purity of
the DNA, and the value for DNA should be ≥1.7–2.0.
Aermo Scientic NanoDrop can be used for the direct
measurement of DNA without diluting the sample.

It is recommended that A correction is used for the
absorbance values. Subtract the A from the A and
A ratios to remove the eects of carryover of the
magnetic beads.

Use these calculations to measure the DNA content:

• Concentration of DNA =
50 µg/ml x (A – A) x dilution factor

• Total amount of DNA =
concentration x volume of sample in ml

• Purity of DNA =
(A – A)/(A – A)

Quantification and

determination of

the purity of DNA

Thermo Fisher Scientific30 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Protocols and Pipetting Instructions

Quantification and determination of the purity of DNA

Thermo Fisher Scientific 31Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Chapter 6

General Information

A reagent must not be used with any kit other than that for
which it is intended. It is strongly recommended that the
volume of reagents in each well or tube is kept within the
limits specied in the KingFisher Flex User Manual or the
KingFisher Duo User Manual to avoid spillover and to keep
performance at the most ecient level.

e KingFisher Magnetic Beads have a tendency to
sediment relatively quickly in the Binding Buer. Once
a premixture of the beads and Binding Buer has been
made, the mixture should be used immediately to avoid
the risk of transferring variable amounts of the beads to
the respective wells or tubes. e amount of the beads in
the wells or tubes aects the yield of the puried DNA.

e binding between the puried DNA and the
KingFisher Magnetic Beads is strong in the presence of
a chaotropic salt, but chaotropic salts are not present in
the Wash Buer 3 and accordingly the binding is weak.
Avoid strong mixing speeds and releasing the magnetic
beads into the Wash Buer 3 in order to minimize the loss
of DNA during the wash step. A short wash and a slow
mixing speed without releasing the magnetic beads into
the buer are recommended.

Reagent specificity

and volumes

Handling of

magnetic beads

Binding and

wash steps

Thermo Fisher Scientific32 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

General Information

Elution step

Carryover of ethanol to the Elution Buer causes impurities
in the Elution Buer and may aect some downstream
applications. To remove traces of ethanol, make sure that
there is a wash step (e.g., washing without releasing the
beads) or the drying step before the elution step is long
enough. ere is a delicate balance between complete
removal of the ethanol and loss of DNA.

e volume of the Elution Buer can be modied
depending on the user requirements concerning
the puried DNA concentration. e nal DNA
concentration in the Elution Buer may increase if the
puried DNA is eluted into a smaller volume of the
buer, but this can slightly reduce the overall DNA yield.
e modications of the elution step must be done in
BindIt Software 3.2 and according to the volume ranges
suitable for the KingFisher instrument. e table below
indicates the available elution volumes of the KingFisher
instruments.

Table 6-1. Available elution volumes of Thermo Scientific KingFisher

instruments

KingFisher instrument Elution volume

KingFisher 20–200 µl

KingFisher mL 50–1000 µl

KingFisher Duo with 12-pin magnet head 30–130 µl

KingFisher Duo with 6-pin magnet head 200–5000 µl

KingFisher Flex with 96 deep well head,
elution in a KingFisher Flex 96 KF plate

50–150 µl

KingFisher Flex with 96 deep well head,
elution in a Microtiter deep well 96 plate

50–1000 µl

KingFisher Flex with 96 well head,
elution in a KingFisher Flex 96 KF plate

20–250 µl

KingFisher Flex with 24 deep well head 200–5000 µl

Elution step

Thermo Fisher Scientific 33Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

General Information

Elution step

To gain a maximal yield of puried DNA, avoid the lowest
permitted volumes of Elution Buer in the KingFisher
instruments. e Elution Buer should completely
cover the KingFisher Magnetic Beads and any possible
magnetic-bead pellet should be completely resuspended.
In addition, the volume of the Elution Buer should
be adequate for ecient mixing of the beads in order to
obtain a maximal release of the puried DNA from the
beads.

Elution can be conducted at room temperature. e elution
step at 72°C in the KingFisher Flex and KingFisher Duo
or at 55°C outside the other KingFisher instruments will,
however, increase the yield by approximately 15–20%.
When using the KingFisher instrument without a heating
option, the Elution Buer can be prewarmed to 55°C
and dispensed into the Elution tubes or wells during an
additional pause in the protocol before the elution step.

If some KingFisher Magnetic Beads remain in the Elution
Buer, centrifuge the Elution plate briey or place it
on a magnet for a few minutes to collect the residual
magnetic beads at the bottom of the well and transfer the
supernatant to a new tube.

RNase treatment is recommended if the purity of the
DNA is not sucient for downstream applications with
the normal protocol.

Thermo Fisher Scientific34 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

General Information

Elution step

Thermo Fisher Scientific 35Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Appendix A

Troubleshooting

Table A-1. Troubleshooting guide

Problem Possible cause and actions

Low DNA yield There should be an adequate volume of the Elution Buffer to cover

the KingFisher Magnetic Beads completely during the elution step.

Do not let the KingFisher Magnetic Beads dry, as this may result in
lower elution efficiency.

Heating during the elution step or using a preheated Elution Buffer
enhances the release of the DNA from the KingFisher Magnetic
Beads and the yield of the purified DNA.

Efficient homogenization and lysis of the plant samples increase the
DNA yield.

Prolonged storage of the sample material may reduce the DNA yield.

If the KingFisher instrument mixes too strongly, it may cause partial
elution of DNA already in the Wash Buffer 3.

Use only Thermo Scientific KingFisher plates, tubes and tip combs
with the KingFisher instruments. Use of products from other
manufacturers may cause unsuitable mixing and affect the yield of
purified DNA.

Low purity Prolonged storage of the sample material may reduce the quality of

the DNA.

Insufficient washing causes impurities in the Elution Buffer.

Carryover of 80% ethanol from the EtOH step of the BindIt protocol
may cause unsatisfactory performance in downstream applications.

Carryover of the KingFisher Magnetic Beads to the Elution Buffer
may affect the A

260/A280

ratio. Make sure that the KingFisher Magnetic
Beads do not affect the measurement by centrifuging the samples
or placing them on a magnet for a few minutes to collect the
residual beads at the bottom of the well. Carryover of the KingFisher
Magnetic Beads does not affect most downstream processes.

Continued

Thermo Fisher Scientific

Troubleshooting

36 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Problem Possible cause and actions

Magnetic particles remaining
in the lysed sample or elution well

Starting material that is too viscose prevents efficient collection of
the KingFisher Magnetic Beads from the lysed sample. The magnetic
rods will not be able to collect all the magnetic beads unless the
viscose samples are diluted before the beginning of the purification
process. The samples can, for example, be diluted into 1 x PBS.
Improper lysis may also cause problems collecting the KingFisher
Magnetic Beads.

If the KingFisher Magnetic Beads are inefficiently collected from
the Elution Buffer, you should add a small amount of detergent
(e.g., 0.02% Tween 20) to improve the results.

Carryover of the KingFisher Magnetic Beads to the Elution Buffer
may affect the A

260/A280

ratio. Refer to "Low purity" on page35.

KingFisher Magnetic Beads that occasionally remain attached to the
tip combs at the end of the process do not affect the DNA yield, as
the DNA has already been released from the KingFisher Magnetic
Beads into the Elution Buffer.

If the KingFisher magnetic particle processor does not work properly,
refer to the relevant user manual of the KingFisher instrument in use.

Continued

Thermo Fisher Scientific 37Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Appendix B

Ordering Information

Table B-1. Thermo Scientific KingFisher Plant DNA Kit

Cat. No. Product Package size

97050196 KingFisher Plant DNA Kit 1 x 96

Table B-2. 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

Table B-3. 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

Thermo Fisher Scientific

Ordering Information

38 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Table B-4. 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 B-5. 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 Scientific Oy
Ratastie 2, P.O. Box 100
FI-01621 Vantaa
Finland

www.thermoscientific.com

N11996