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Thermo Fisher Scientific

21

Thermo Scientific KingFisher Plant DNA Kit Instruction Manual

Chapter 5

Protocols and Pipetting

Instructions

Before beginning the DNA purification protocol,

carefully read through the

Thermo Scientific KingFisher

Flex User Manual

(Cat. No. N07669), the

Thermo

Scientific KingFisher Duo User Manual

(Cat. No. N12420)

and the

Thermo Scientific 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 briefly. The KingFisher Magnetic

Beads have a tendency to sediment relatively quickly in

the Binding Buffer. Once a premixture of the beads and

the Binding Buffer 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 fifth 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

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Содержание KingFisher Plant DNA Kit

Страница 1: ...Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Rev 1 1 ...

Страница 2: ......

Страница 3: ...Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Rev 1 1 Cat no N11996 ...

Страница 4: ...oduct will operate substantially in conformance with Thermo Fisher Scientific s published specifications 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 SCIE...

Страница 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 purification 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 purification from 400 µl...

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

Страница 7: ...ysis 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 The KingFisher Plant DNA Kit Cat No 97050196 is intended for the purification of 96 samples using the Thermo Scientific KingFisher Flex with a 96 deep well head o...

Страница 8: ...6 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 ...

Страница 9: ... 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 KingFishe...

Страница 10: ...Thermo Fisher Scientific 10 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Kit Content ...

Страница 11: ...ss requires no phenol chloroform extraction or alcohol precipitation and needs very little hands on time The reagents and specific plastic consumables are designed to work with the KingFisher Flex or KingFisher Duo magnetic particle processors as part of an integrated system The KingFisher Plant DNA Kit is only intended for research use not for clinical or diagnostic use The user is responsible fo...

Страница 12: ...pid rinse in the third Wash Buffer removes the residual ethanol and considerably improves the purity of the DNA High quality DNA is eluted into the Elution Buffer 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 The KingFisher Plant DNA Kit is designed for rapid automated pr...

Страница 13: ...even instability in the KingFisher instrument The 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 Thermo Scientific Multidrop Combi and or the Ther...

Страница 14: ...er 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 skir...

Страница 15: ...be 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 The 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 pro...

Страница 16: ...Thermo Fisher Scientific 16 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Product Description KingFisher magnetic particle processors ...

Страница 17: ...ble 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 Sod...

Страница 18: ...Thermo Fisher Scientific 18 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Safety Information ...

Страница 19: ...he 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 The buffers are ready for use To prepare the RNase A enzyme for the Thermo Scientific 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 ...

Страница 20: ...Thermo Fisher Scientific 20 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Storage Conditions and Preparation of the Working Solution Preparation of the RNase A working solution ...

Страница 21: ...s 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 briefly The KingFisher Magnetic Beads have a tendency to sediment relatively quickly in the Binding Buffer Once a premixture of the beads and the Binding Buffer has been made the mixtu...

Страница 22: ...ling 96 samples simultaneously The 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 Buffer to a homogenized sample and mix for 30 seconds If RNase A treatment is needed to reduce the amount of RNA ...

Страница 23: ...er 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 w...

Страница 24: ...er 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 The 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...

Страница 25: ...icrotiter 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 Buff...

Страница 26: ...lution 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 ...

Страница 27: ... 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...

Страница 28: ...ed nucleic acid is eluted into a smaller volume of the buffer 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...

Страница 29: ...80 indicates the purity of the DNA and the value for DNA should be 1 7 2 0 A Thermo Scientific NanoDrop can be used for the direct measurement of DNA without diluting the sample It is recommended that A320 correction is used for the absorbance values Subtract the A320 from the A260 and A280 ratios to remove the effects of carryover of the magnetic beads Use these calculations to measure the DNA co...

Страница 30: ...Thermo Fisher Scientific 30 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual Protocols and Pipetting Instructions Quantification and determination of the purity of DNA ...

Страница 31: ...Binding Buffer 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 The amount of the beads in the wells or tubes affects the yield of the purified DNA The binding between the purified DNA and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt but chaotropic salts are not pres...

Страница 32: ... into a smaller volume of the buffer but this can slightly reduce the overall DNA yield The modifications of the elution step must be done in BindIt Software 3 2 and according to the volume ranges suitable for the KingFisher instrument The table below indicates the available elution volumes of the KingFisher instruments Table 6 1 Available elution volumes of Thermo Scientific KingFisher instrument...

Страница 33: ...t room temperature The 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 Buffer can be prewarmed to 55 C and dispensed into the Elution tubes or wells during an additional pause in the protocol before the e...

Страница 34: ...Thermo Fisher Scientific 34 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual General Information Elution step ...

Страница 35: ...nt 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 cau...

Страница 36: ...er 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 A260 A280 ratio Refer to Low purity on page 35 KingFisher Magnetic Beads that...

Страница 37: ...b 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 1...

Страница 38: ...002131 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 b...

Страница 39: ......

Страница 40: ...Thermo Fisher Scientific Oy Ratastie 2 P O Box 100 FI 01621 Vantaa Finland www thermoscientific com N11996 ...

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