Automated purification with KingFisher® instruments
Contents
1. KingFisher Software protocol for MACHEREY NAGEL NucleoMag 96 Tissue kit can be downloaded from the website www thermo com kingfisher First you have to save the file NucleoMagTissueKF96 to your computer 1 Open KingFisher Software 2 Select Protocol Import Export data 3 Click Read file 4 Select the database KF2 by browsing in the Open dialog and click Open 5 Select the protocol s you wish to import from the Protocols in file list Use the SHIFT key together with the mouse button to select protocols between two clicked protocols and the CTRL key to select only the clicked protocols 6 Tick Update existing if you wish to overwrite the protocols with identical protocol name s in the target database 7 Click Import If there are protocols with identical names and you have not ticked the Update existing tick box you will be prompted to change the name of the protocol that is being imported Type in a new name and click OK o Note Check that the name of the protocol does not exceed 17 characters You will receive a message stating whether the database updating procedure was successful or not Sample preparation e KingFisher DNA protocol NucleoMag TissueKF96 is designed to purify DNA from tissue samples cells or bacteria e Use Microtiter 96 DW plate Catalog No 95040450 KingFisher 96 tip comp for DW magnets Catalog No 97002534 and KingFisher 96 KF plate Catalog No 97002540 with NucleoMag 96 T2. applications Contact information Thermo Fisher SCIENTIFIC Ratastie 2 P O Box 100 FIN 01621 Vantaa Finland Tel 358 9 329100 Fax 358 9 32910415 www thermo com MACHEREY NAGEL Neumann Neander Str 6 8 D 52355 D ren Germany T 49 0 2421 969 0 F 49 0 2421 969 199 www mn net com email sales mn net com 3 Any steps of the protocol e g sample incubation and elution times and the reagent volumes can be modified with KingFisher software 4 Tip comb was forgotten gt Clean the magnetic rods using a soft cloth or tissue paper soaked in mild detergent solution soap or alcohol 5 The processor is not working properly gt Refer to Kingfisher User Manual
3. Thermo Fisher SCIENTIFIC MACHEREY NAGEL USER GUIDE for Automated purification of DNA from cells or tissue samples with KingFisher 96 KingFisher mL instrument and MACHEREY NAGEL NucleoMag 96 Tissue kit 10 11 2006 ThermoFisher SCIENTIFIC Descripition Purification of DNA from cells or tissue e g mouse tails samples with MACHEREY NAGEL s NucleoMag 96 Tissue kit can easily be automated using KingFisher instruments Thermo Electron Corporation The KingFisher platforms utilize patented technology where magnetic rods move particles through the processing steps KingFisher 96 instrument operates on microplates and can process up to 96 samples per run whereas KingFisher mL can handle 15 samples per run With both instruments the volumes handled can be up to 1 ml Typically DNA isolation from 20 mg of mouse tail clippings using KingFisher instruments results up to 20 ug DNA Generally DNA yields vary according to sample type and storage conditions The protocol described here is designed for general use and can be modified according to customer individual needs using KingFisher Software provided with the instrument Important notes e See MACHEREY NAGEL NucleoMag 96 Tissue kit user manual for reagent storage product use limitations safety information etc e Resuspend magnetic beads DNA Binding Beads thoroughly before use MACHEREY NAGEL KingFisher 96 protocol Importing protocols from the web
4. g 6 Beads are discarded into plate 3 ThermoFisher SCIENTIFIC KingFisher mL protocol Importing protocol from the web KingFisher mL Software protocol for NucleoMag 96 Tissue kit can also be downloaded from the website www thermo com kingfisher Save the file NucleoMagTissueKF_mL to your computer Sample preparation e KingFisher mL DNA protocol NucleoMag TissueKFmL is designed to purify DNA from tissue samples cells or bacteria Use KingFisher mL tubestrips and tip combs Catalog No 97002141 with NucleoMagTissueKFmL protocol e Add sample and other reagents supplied by NucleoMag 96 Tissue kit to KingFisher mL tubestrips according to table 2 and instructions below KingFisher mL process Table 2 Pipetting instructions for KingFisher mL and NucleoMag 96 Tissue protocol Sample Reagent Tube volume Lysed Sample 225 ul A Magnetic Beads 24 ul B Wash buffermes Soop D elution buffer mes 800p Place an appropriate number of tube strips needed for the samples one tube strip per sample into removable tube strip tray 2 Add 225 ul of lysed sample 24 ul of Magnetic Beads and 360 ul of Binding Buffer MB2 to tube strip A 3 Add 600 ul of Wash Buffer MB3 and Wash Buffer MB4 to tube strip B and C respectively 4 Add 800 ul of Wash Buffer MB5 to tube strip D 5 Add 100 p of Elution Buffer MB6 to tube strip E MACHEREY NAGEL 6 Close the front lid and start the process by selecting in
5. issue protocol e Add sample and other reagents except Elution Buffer supplied by NucleoMag 96 Tissue kit to KingFisher deepwell microplate according to table 1 and instructions below Add the Elution Buffer to KingFisher 96 plate and start the NucleoMagTissue_KF96 process ThermoFisher SCIENTIFIC KingFisher 96 process Table 1 Pipetting instructions for KingFisher 96 and NucleoMag 96 Tissue protocol Sample Reagent a re OE Magnetic Beads added after lysis 24 ul A 1 step Binding buffer MB2 added after lysis a step 360 ul B 5 Elution bufferMB6 100 yl A Microtiter 96 DW plate B KingFisher 96 KF plate 1 Add 205 ul of buffer T1 and 20 ul of proteinase K solution to sample Performe lysis at 56 C for 1h 16h depending on samples Following lysis load lysed sample 24 ul of resuspended Magnetic Beads and 360 ul Binding Buffer MB2 to plate 1 Add 600 ul of Wash Buffer MB3 to plate 2 Add 600 ul of Wash Buffer MB4 to plate 3 Add 800 ul of Wash Buffer MB5 to plate 4 Add 100 ul of Elution Buffer MB6 to plate 5 Combine the tip comb and The KingFisher plate See KingFisher 96 User manual Select the NucleoMag 96 Tissue protocol using arrow keys and press START button 8 Load the plates according to protocol request and press START after every plate to confirm the action 9 Note Confirm that the plates are placed in correct orientation A1 well to be pointed to upper right corner
6. o not incubate beads in this buffer for more than 2 min as this buffer is water based and might elute the DNA already 2 Low purity gt Poor sample quality e Check sample storage Samples can be stored at 2 8 C for some days Freeze samples if stored for longer periods gt Insufficient washing procedure e Use only recommended Deep well blocks see KingFisher User Manual gt Carry over of ethanol from wash buffer MB4 MACHEREY NAGEL Ordering Information Product Description no 540 05 00 KingFisher 96 110V 240V Magnetic particle processor 24073430 Magnet head for Deep Well plate KingFisher 96 tip comb for DW ml UES magnets 10 x 10 pcs box KingFisher 96 plate 200 ul 48 plates box Deep Well 96 plate V bottom Polypropylene KingFisher mL 110 240 V Magnetic particle processor KingFisher mL Combi 60 tubes and tips for 60 samples KingFisher mL Combi 240 TOUSA tubes and tips for 240 samples 7002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 900 pcs 20X45 MACHEREY NAGEL 97002540 95040450 540 00 50 97002131 744 300 1 NucleoMag 96 Tissue kit 1x96 preps 744 300 4 NucleoMag 96 Tissue kit 4x96 preps 744 300 24 NucleoMag 96 Tissue kit 24x96 preps e Increase time for washing step with buffer MB5 to 2 min Alternatively add additional air dry step 5 min Be sure to remove all of the ethanolic Wash Buffer MB4 as residual ethanol interferes with downstream
7. of the plate holder in turntable A1 row of the plate is then always located in the inner circle of the turntable 10 The purification protocol will start when the last plate is loaded and START button is pressed 11 After lysis step add 24 ul of resuspended Magnetic Beads and 360 ul of Binding Buffer MB2 to plate 1 Beads and Binding buffer may be premixed before addition to plate 1 12 After the purification process is completed the plates are removed according to instructions shown in instrument screen Oak WNY N MACHEREY NAGEL Press START after each plate removal to confirm the action 13 When the last plate is removed text End_of _run will appear Press STOP to complete the run Description of NucleoMag 96 Tissue protocol with KingFisher 96 1 Samples are lysed with buffer T1 and proteinase K for 1 16 h at 56 C Lysis incubation time depends on sample type Refer NucleoMag 96 Tissue kit protocol for details Following Lysis all further steps are done on the KingFisher 96 instrument 2 Lysed samples are incubated first with magnetic beads and Binding Buffer MB2 in plate 1 for 5 minutes Magnetic bead DNA complexes are formed 3 Magnetic beads are washed with Wash Buffer MB3 and MB4 in plates 2 and 3 respectively 4 A final short wash step of magnetic beads with Wash buffer MB5 in plate 4 removes ethanol from previous wash buffers 5 DNA is released to Elution Buffer in plate 5 for 10 minutes with heatin
8. tended protocol NucleoMagTissue_KFmL using arrow keys and by pressing START 7 Remove the tube strip tray from the KingFisher mL after program has completed Description of NucleoMag 96 Tissue protocol with KingFisher mL 1 Samples are lysed with buffer T1 and proteinase K for 1 16 h at 56 C Lysis incubation time depends on sample type Refer NucleoMag 96 Tissue kit protocol for details Following lysis all further steps are done on the KingFisher ml instrument 2 Lysed samples are incubated first with magnetic beads and Binding Buffer MB2 for 5 minutes in the tube strips A Magnetic bead DNA complexes are formed 3 Magnetic beads are washed with Wash Buffer MB3 and MB4 in tube strips B and C respectively 4 A final short wash step of magnetic beads with Wash buffer MB5 in tube strip D removes ethanol from previous wash buffers 5 Sample is released to Elution Buffer MB6 in tube strip E Optionally The sample is then moved manually to 1 5 ml reaction tube and incubated in a heat block for 10 minutes to release the DNA The sample is then moved back to tube strip E 6 Beads are discarded into tube strip C ThermoFisher SCIENTIFIC Trouble shooting 1 Low yield gt Elution buffer volume insufficient e Beads pellet must be covered completely with elution buffer gt Partial elution in Wash Buffer MB5 already e Keep the beads on the magnet while washing in Wash Buffer MB5 Do not resuspend beads in this buffer and d
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