Genomic DNA from plant
Contents
1. Natriumperchlorat 5 20 Ethanol 20 35 RNase A RNase A Iyophilized x Xn R 42 43 S 22 24 RNase A lyophilisiert Risk phrases R10 Flammable Entz ndlich R 11 Highly Flammable Leichtentz ndlich R 36 Irritating to eyes Reizt die Augen R 42 43 May cause sensitization by inhalation and contact with skin Sensibilisierung durch Einatmen und Hautkontaktm glich R 67 Vapours may cause drowsiness and dizziness D mpfe k nnen Schl frigkeit und Benommenheit verursachen Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not necessary if quantity per bottle below 25g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 02 2012 Rev 02 11 Genomic DNA from plant Safety phrases S7 Keep container tightly closed Beh lter dicht geschlossen halten S16 Keep away from sources if ignition No smoking Von Z ndquellen fernhalten Nicht rauchen 522 Do not breathe dust Staub nicht einatmen 524 Avoid contact with the skin Ber hrung mit der Haut vermeiden S26 In case of contact with eyes rinse immediately with plenty o2. Genomic DNA from plant User manual NucleoMag 96 Plant February 2012 Rev 02 MACHEREY NAGEL MN Genomic DNA from plant Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 4 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Magnetic separation systems 6 2 4 Adjusting the shaker settings 7 2 5 Handling of beads 8 2 6 Storage and homogenization of samples 9 2 7 Elution procedures 9 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 4 1 Risk and safety phrases 11 4 2 GHS classification 12 5 Protocol for the isolation of genomic DNA from plant samples 14 6 Appendix 19 6 1 Troubleshooting 19 6 2 Ordering information 21 6 3 Product use restriction warranty 22 MACHEREY NAGEL 02 2012 Rev 02 3 Genomic DNA from plant 1 Components 1 1 Kit contents NucleoMag 96 Plant 1x 96 preps 4 x 96 preps 24 x 96 preps REF 744400 1 744400 4 744400 24 NucleoMag C Beads 3mL 12mL 72 mL Lysis Buffer MC1 60 mL 240 mL 3 x 480 mL Binding Buffer MC2 2 x 25 mL 8x 25 mL 3 x 400 mL Wash Buffer MC3 75 mL 2x 150 mL 2 x 900 mL Wash Buffer MC4 75 mL 2x 150 mL 2 x 900 mL Wash Buffer MC5 75 mL 2x 150 mL 2 x 900 mL Elution Buffer MC6 25 mL 100 mL 600 mL RNase A lyophilized 15 mg 2x30 mg 12 x 30 mg Elution Plate U bottom including Self adhering 1 4 24 Foil User manual 1 1 1 1 2 Reagents
3. Plant tissue is extracted with CTAB Lysis Buffer MC1 Adjusting the binding conditions of nucleic acid with Binding Buffer MC2 and addition of paramagnetic beads can be carried out simultaneously After magnetic separation and removal of supernatant the paramagnetic beads are washed with Wash Buffers MC3 MC4 and 80 ethanol to remove contaminants and salt There is no need for a drying step as ethanol from previous wash steps is removed by Wash Buffer MC5 Finally highly purified DNA is eluted with low salt Elution Buffer MC6 and can directly be used for downstream applications The NucleoMag 96 Plant kit can be used either manually or automated on standard liquid handling instruments 2 2 Kit specifications NucleoMag 96 Plant is designed for rapid manual and automated small scale preparation of DNA from plant samples The kit is designed for use with NucleoMag SEP magnetic separator plate see ordering information or other magnetic separation systems see section 2 3 Manual time for the preparation of 96 samples is about 120 minutes The purified DNA can be used directly as template for qPCR next generation sequencing or any kind of enzymatic reactions NucleoMag 96 Plant allows easy automation on common liquid handling instruments or automated magnetic separators The actual processing time depends on the configuration of the instrument and the magnetic separation system used Typically 96 samples can be purified in less than
4. 120 minutes using the NucleoMag SEP on the automation platform 2 3 Magnetic separation systems For use of NucleoMag 96 Plant the use of the magnetic separator NucleoMag SEP is recommended Separation is carried out in a Square well Block see ordering information The kit can also be used with other common separators Magnetic separator Separation plate or tube NucleoMag SEP MN REF 744900 Square well Block MN REF 740481 Tecan Te MagS 1 5 mL tubes without lid Sarstedt 6 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant Static magnetic pins Separators with static magnetic pins for example NucleoMag SEP for manual use and for use on liquid handling workstations This type of separator is recommended in combination with a suitable microplate shaker for optimal resuspension of the beads during the washing and elution steps Alternatively beads can be resuspended in the buffer by pipetting up and down several times For fully automated use on liquid handling workstations a gripper tool is required the plate is transferred to the magnetic separator for separation of the beads and transferred to the shaker module for resuspension of the beads Movable magnetic systems Separators with moving magnetic pins Magnetic pins rods are moved from one side of the well to the other and vice versa Beads follow this movement and are thus pulled through the buffer during the wash and elution steps Separa
5. or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 02 2012 Rev 02 23 Genomic DNA from plant components not manufactured by M
6. pins e g NucleoMag SEP and suitable plate shakers see section 2 3 It is recommended using a Square well Block for separation see section 1 2 Alternatively isolation of DNA can be performed in reaction tubes with suitable magnetic separators This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments Before starting the preparation Check if RNase A was prepared according to section 3 1 Homogenize and lyse sample material Homogenize about 20 50 mg fresh or lt 10 mg lyophilized plant tissue for example using mictrotube strips in a mixer mill and add 500 uL Buffer MC1 Do not moisten the rim Close the individual wells with cap strips Mix by vigorous shaking for 15 30 s Spin briefly for 30 s at 1 500 x g to collect any sample from the cap strips Incubate the closed strips at 56 C for 30 min Optional If samples contain large amounts of RNA we recommend the addition of 10 uL RNase A solution stock solution 12 mg mL to the MC1 lysis mixture 2 Clear lysates Centrifuge the samples for 20 min at a full speed 5 600 6 000 x g Remove cap strips Transfer 400 uL of the cleared lysate equilibrated to room temperature to a Square well Block Do not moisten the rims of the well Note See recommendations for suitable plates or tubes and compatible magnetic separators section 1 2 3 Bind DNA to NucleoMag C Beads Add 30 pL of NucleoMag C Beads and 400 u
7. ACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authori
8. L Buffer MC2 to each well of the Square well Block Mix by pipetting up and down 6 times and shake for 5 min at room temperature Alternatively when processing the kit without a shaker pipette up and down 10 times and incubate for 5 min at room temperature Note NucleoMag C Beads and Buffer MC2 can be premixed For 96 samples mix at least 2880 uL of NucleoMag C Beads with 38 4 mL of Buffer MC2 mix by vortexing Use 430 uL of the suspension per well Be sure to resuspend the NucleoMag C Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has been formed MACHEREY NAGEL 02 2012 Rev 02 17 NucleoMag 96 Plant Separate the magnetic beads against the side of the wells by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipetting Note Do not disturb the attracted beads while aspirating the supernatant The magnetic pellet is not visible in this step Remove supernatant from the opposite side of the well Wash with MC3 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 uL Buffer MC3 to each well and resuspend the beads by shaking until the beads are resuspended completely 5 min Alternatively resuspend beads completely by repeated pipetting up and down 15 times Separate the magnetic beads by
9. ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism
10. RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 4 Wash with MC3 Remove Square well Block from NucleoMag SEP 600 pL MC3 14 MACHEREY NAGEL 02 2012 Rev 02 NucleoMag 96 Plant Resuspend Shake 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 5 Wash with MC4 Remove Square well Block from NucleoMag SEP 600 pL MC4 Resuspend Shake 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation Wash with Remove Square well Block 80 ethanol from NucleoMag SEP 600 uL 80 ethanol Resuspend Shake 5 min at RT Optional Mix by pipetting up and down Remove supernatant MACHEREY NAGEL 02 2012 Rev 02 15 NucleoMag 96 Plant 7 8 Wash with MC5 Elute DNA Leave Square well Block on NucleoMag SEP 600 pL MC5 Incubate for 45 60 s Note Do not resuspend the beads in Buffer MC5 Remove supernatant Remove Square well Block from NucleoMag SEP 50 200 pL MC6 Optional Elute at 55 C Shake 5 min at RT Optional Mix by pipetting up and down Separate 2 min and transfer DNA into elution plate tubes 16 MACHEREY NAGEL 02 2012 Rev 02 NucleoMag 96 Plant Detailed protocol This protocol is designed for magnetic separators with static
11. consumables and equipment to be supplied by user Reagents 80 ethanol For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant Equipment Consumables Product REF Pack of Magnetic separation system 744900 1 e g NucleoMag SEP see section 2 3 Separation plate for magnetic beads separation 740481 4 e g Square well Block 96 well block with 740481 24 24 2 1 mL square wells Lysis tubes for incubation of samples ana yas 740477 4 sets e g Rack of Tubes Strips 1 set consists 740477 24 24 sets of 1 Rack 12 Strips with 8 tubes 1 2 mL wells each and 12 Cap Strips Elution plate for collecting purified nucleic acids 740486 24 24 e g Elution Plate U bottom 96 well 0 3 mL microtiterplate with 300 uL u bottom wells e g Elution Plate Flat bottom 96 well 0 3 mL microtiterplate with 300 uL flat bottom wells 740673 20 For use of kit on KingFisher 96 instrument e g KingFisher 96 Accessory Kit B Square well Blocks Deep well tip combs 744951 Elution Plates for 4 x 96 NucleoMag 96 Plant preps using KingFisher 96 platform 1 set MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant 2 Product description 2 1 The basic principle The NucleoMag 96 Plant procedure is based on reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions
12. d pellet is clearly visible Gently add 600 uL Buffer MC5 to each well and incubate for 45 60 s while the beads are still attracted to magnets Then aspirate and discard the supernatant Note Do not resuspend the beads in Wash Buffer MC5 This step is to remove traces of ethanol and eliminates a drying step Elution Remove the Square well Block from the NucleoMag SEP magnetic separator Add desired volume of Buffer MC6 50 200 pL to each well of the Square well Block and resuspend the beads by shaking 5 10 min at 56 C Alternatively resuspend beads completely by repeated pipetting up and down and incubate for 5 10 min at 56 C Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Transfer the supernatant containing the purified genomic DNA to the Elution Plate Note Yield can be increased by 15 20 by using pre warmed elution buffer 55 C or by incubating the bead elution buffer suspension at 55 C for 10 min MACHEREY NAGEL 02 2012 Rev 02 19 Genomic DNA from plant 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor DNA yield Elution buffer volume insufficient Beads pellet must be covered completely with elution buffer Insufficient performance of elution buffer during elution step Remove residual buffers during the separation steps comp
13. f water and seek medical advice Bei Ber hrung mit den Augen sofort gr ndlich mit Wasser absp len und Arzt konsultieren 539 Wear eye face protection Schutzbrille Gesichtsschutz tragen 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 125 mL or 125 9 Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze MC2 Isopropanol 50 100 Danger 225 319 210 233 280 Isopropanol 50 100 Gefahr 336 305 351 338 337 313 403 235 MC3 MC4 Sodium perchlorate Warning 226 210 233 5 20 ethanol 20 35 403 235 Natriumperchlorat 5 20 Achtung Ethanol 20 35 RNase A RNase A Iyophilized Danger 317 334 261 280 RNase A Iyophilisiert Gefahr 302 352 304 341 333 313 3424311 363 12 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant Hazard phrases H 225 H 226 H 317 H 319 H 334 H 336 Highly flammable liquid and vapour Fl ssigkeit und Dampf leicht entz ndbar Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen Causes serious eye irritation Verursacht schwere Augenreizung May cause allergy or asthma symptoms or breathing difficulties if
14. he Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doc tor physician Bei Symptomen der Atemwege Giftinformationszentrum oder Arzt anrufen Get medical advice attention Bei anhaltender Augenreizung Arztliche Rat einholen rztliche Hilfe hinzuziehen Store in a well ventilated place Keep cool Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 02 2012 Rev 02 13 NucleoMag 96 Plant 5 Protocol for the isolation of genomic DNA from plant samples Protocol at a glance For additional equipment and hardware requirements refer to section 1 2 and 2 3 respectively For detailed information on each step see page 17 Before starting the preparation Check if RNase A was prepared according to section 3 1 Homogenize and 500 pL MC1 lyse plant sample material 20 50 mg Mix 56 C 30 min 2 Clear lysates by 5 600 x g centrifugation transfer 20 min 400 uL of cleared lysate to a Square well Block for further processing 400 uL cleared lysate 3 Bind DNA to NucleoMag C Beads 30 uL NucleoMag C Beads 400 pL MC2 Mix by shaking for 5 min at
15. inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen May cause drowsiness or dizziness Kann Schl frigkeit und Benommenheit verursachen Precaution phrases P 210 P 233 P 261 P 280 P 302 352 P 304 341 P 305 351 313 P 333 313 P 342 311 P 337 313 P 403 235 P 363 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme hei en Oberfl chen fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen IF INHALED If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing Bei Einatmen Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len IF skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztlic
16. ions and preparation of working solutions Attention Buffers MC3 and MC4 contain chaotropic salt Buffer MC2 is highly flammable and irritant Wear gloves and goggles Storage conditions All components of the NucleoMag 96 Plant kit should be stored at room temperature 18 25 C and are stable for up to one year All buffers are delivered ready to use Before starting any NucleoMag 96 Plant protocol prepare the following RNase A Before first use add the indicated volume of water to each vial of the lyophilized RNase A Store RNase A at 4 C 80 ethanol Use molecular biology grade ethanol dilute with appropriate water to 80 NucleoMag 96 Plant 1 x 96 preps 4x 96 preps 24 x 96 preps REF 744400 1 744400 4 744400 24 RNase A 15 mg 2 x 30 mg 12 x 30 mg lyophilized Add 1 25 mL water Add 2 5 mL water Add 2 5 mL water to each vial to each vial 10 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant 4 Safety instructions The following components of the NucleoMag 96 Plant kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol MC2 Isopropanol 50 100 Fe R 11 36 67 S 7 16 Isopropanol 50 100 24 26 39 MC3 MC4 Sodium perchlorate 5 20 ii R 10 S 16 ethanol 20 35
17. letely Remaining buffers decrease efficiency of following wash steps and elution step Beads dried out Do not let the beads dry as this might result in lower elution efficiencies Partial elution in Wash Buffer MC5 already Keep the beads on the magnet while dispensing Wash Buffer MC5 Do not resuspend beads in this buffer and do not incubate beads in this buffer for more than 2 min as this buffer is water based and might elute the DNA already Aspiration of attracted bead pellet Do not disturb the attracted beads while aspirating the supernatant especially when the magnetic pellet is not visible in the lysate Incubation after dispensing beads to lysate Mix immediately after dispensing NucleoMag C Beads Buffer MC2 to the lysate Insufficient washing procedure Use only the appropriate combinations of separator and Low purity plate for example Square well Block in combination with NucleoMag SEP 20 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant Problem Possible cause and suggestions Carry over of ethanol from 80 ethanol wash solution Suboptimal cf Be sure to remove all of the ethanolic wash solution as pe ormance residual ethanol interferes with downstream applications of DNA in downstream Fow oun applications OW peny See above Carry over of beads Time for magnetic separation too short Increase separation time to allow the beads to be completely attracted to
18. mogenizing samples by VA steel beads diameter 7 mm Put 4 5 beads and plant material together into a 15 mL plastic tube Falcon chill the tube in liquid nitrogen and vortex for about 30 seconds e g with a Multi Pulse Vortexer contact Sch tt Labortechnik GmbH Postfach 3454 D 37024 G ttingen Germany Repeat this chilling and vortexing procedure until the entire plant material is ground to a powder Chill the tube once more and remove the beads by rolling them out gently or with a magnet Keep the material frozen throughout the whole homogenization procedure Do not add nitrogen to the tube This leads to sticking and loss of plant material attached to the beads 2 7 Elution procedures Purified DNA can be eluted directly with the supplied Elution Buffer MC6 Elution can be carried out in a volume of gt 50 uL It is essential to cover the NucleoMag Beads completely with elution buffer during the elution step The volume of dispensed elution buffer depends on the magnetic separation system e g the position of the pellet inside the separation plate For efficient elution the magnetic bead pellet should be resuspended completely in the elution buffer For some separators higher elution volumes might be necessary to cover the whole pellet Elution is possible at room temperature Yield can be increased by 15 20 if elution is performed at 55 C MACHEREY NAGEL 02 2012 Rev 02 9 Genomic DNA from plant 3 Storage condit
19. or operation 24 MACHEREY NAGEL 02 2012 Rev 02
20. placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnet Remove and discard supernatant by pipetting Wash with MC4 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 uL Buffer MC4 to each well and resuspend the beads by shaking until the beads are resuspended completely 5 min Alternatively resuspend beads completely by repeated pipetting up and down 15 times Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnet Remove and discard supernatant by pipetting Wash with 80 ethanol Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 uL 80 ethanol to each well and resuspend the beads by shaking until the beads are resuspended completely 5 min Alternatively resuspend beads completely by repeated pipetting up and down 15 times Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnet Remove and discard supernatant by pipetting 18 MACHEREY NAGEL 02 2012 Rev 02 NucleoMag 96 Plant Wash with MC5 Leave the Square well Block on the NucleoMag SEP magnetic separator Note Supernatant is colorless magnetic bea
21. the magnetic pins before aspirating any liquid from the well Aspiration speed too high elution step High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step Cross contamination Contamination of the rims Do not moisten the rims of the Square well Block when transferring the plant lysate If the rim of the wells is contaminated seal the Square well Block with Self adhering PE Foil see ordering information before starting the shaker MACHEREY NAGEL 02 2012 Rev 02 21 Genomic DNA from plant 6 2 Ordering information Product REF Pack of NucleoMag 96 Plant 744400 1 1 x 96 preps 744400 4 4 x 96 preps 744400 24 24 x 96 preps NucleoMag SEP 744900 1 Square well Blocks 740481 4 740481 24 24 Self adhering PE Foil 740676 50 sheets Rack of Tube Strips 740477 4 sets set consists of 1 Rack 12 Tube Strips 740477 24 24 sets with 8 tubes each and 12 Cap Strips Cap Strips 740638 30 strips KingFisher 96 Accessory Kit B 744951 1 set set consists of Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag 96 Plant preps using KingFisher 96 platform Visit www mn net com for more detailed product information 22 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant 6 3 Product use restriction warranty NucleoMag 96 Plant kit components are intended developed designed and sold FOR RESEARCH PURPOSES
22. ting up and down however is more efficient than mixing by a shaker or magnetic mix Resuspension Number of tips efficiency needed Magnetic mix Low Shaker Low Pipetting High acceptable good excellent 8 channel pipetting device 8 MACHEREY NAGEL 02 2012 Rev 02 Genomic DNA from plant 2 6 Storage and homogenization of samples We recommend the use of young plant samples and to keep plants for about 12 hin the dark before collecting samples in order to reduce polysaccharide content Plant samples can be stored frozen under ethanol or lyophilized In many cases lyophilized dried material can be easier processed and gives higher yield If using dried samples reduce the amount of starting material by the factor 5 e g use 10 mg dried plant leaves instead of 50 mg fresh weight As plant tissue is very robust the lysis procedure is most effective with well homogenized powdered samples Suitable methods include grinding with pestle and mortar in the presence of liquid nitrogen or using steel beads We also recommend the use of other commercial homogenizers bead mills etc Methods to homogenize samples Commercial homogenizers for example Crush Express for 96 well homogenization contact Saaten Union Resistenzlabor GmbH D 33818 Leopoldsh he Tissue Striker www KisanBiotech com or Geno Grinder 2000 www spexcsp com or for Germany www c3 analysentechnik de Ho
23. tion takes place when the system stops Automated separators Separators with moving magnets Magnetic beads are transferred into suitable plates or tubes Beads are resuspended from the rod covered magnets Following binding washing or elution beads are collected again with the rod covered magnets and transferred to the next plate or tube 2 4 Adjusting the shaker settings When using a plate shaker for the washing and elution steps the speed settings have to be adjusted carefully for each specific separation plate and shaker to prevent cross contamination from well to well Proceed as follows Adjusting shaker speed for binding and wash steps Load 1000 uL for checking the settings for the binding step or 600 uL for checking the settings for the washing steps dyed water to the wells of the separation plate Place the plate on the shaker and start shaking with a moderate speed setting for 30 seconds Turn off the shaker and check the plate surface for small droplets of dyed water Increase speed setting shake for an additional 30 seconds and check the plate surface for droplets again Continue increasing the speed setting until you observe droplets on top of the separation plate Reduce speed setting check again and use this setting for the washing step Adjusting shaker speed for the elution step Load 100 uL dyed water to the wells of the collection plate and proceed as described above MACHEREY NAGEL 02 2012 Re
24. v 02 T Genomic DNA from plant 2 5 Handling of beads Distribution of beads A homogeneous distribution of the magnetic beads to the individual wells of the separation plate is essential for a high well to well consistency Therefore before distributing the beads make sure that the beads are completely resuspended Shake the storage bottle well or place it on a vortexer shortly Premixing magnetic beads with the binding buffer allows easier homogenous distribution of the beads to the individual wells of the separation plate During automation a premix step before aspirating the beads binding buffer mixture from the reservoir is recommended to keep the beads resuspended Magnetic separation time Attraction of the magnetic beads to the magnetic pins depends on the magnetic strength of the magnetic pins the selected separation plate distance of the separation plate from the magnetic pins and the volume to be processed The individual times for complete attraction of the beads to the magnetic pins should be checked and adjusted on each system It is recommended using the separation plates or tubes specified by the supplier of the magnetic separator Washing the beads Washing the beads can be achieved by shaking or mixing In contrast to mixing by pipetting up and down mixing by shaker or magnetic mixing allows simultaneous mixing of all samples This reduces the time and number of tips needed for the preparation Resuspension by pipet
25. zed officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks KingFisher is a registered trademark of Thermo Fisher Scientific NucleoMag is a registered trademark of MACHEREY NAGEL GmbH amp Co KG Te MagS is a trademark of Tecan Group Ltd Switzerland All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency
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