Genomic DNA from Plant - Lab Supplies Scientific
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1. aspirating any liquid from the well Aspiration speed too high elution step e High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step Contamination of the rims e 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 05 2005 Rev 01 Genomic DNA from Plant 6 2 Ordering information Product Cat No Pack of NucleoMag 96 Plant 744 400 1 1 x 96 NucleoMag 96 Plant 744 400 4 4 x 96 NucleoMag 96 Plant 744 400 24 24 x 96 NucleoMag SEP 744 900 1 Square well Blocks 740 670 20 Self adhering PE foil 740 676 50 sheets MN Tube Strips 740 637 5 sets Cap Strips 740 638 30 strips 6 3 Product use restriction warranty NucleoMag 96 Plant kit components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoMag 96 Plant kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation2. 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 MC7 lysis mixture 2 Clear lysate Centrifuge the samples for 20 min at a full speed 5 600 6 000 x g Remove cap strips 3 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 2 3 4 Add 400 ul Binding Buffer MC2 and 30 ul of NucleoMag Plant Beads to each well of the Square well Block Mix immediately by shaking 5 min at room temperature Alternatively pipette up and down 10 times and incubate 5 min at room temperature Note NucleoMag Plant Beads and Binding Buffer MC2 can be premixed For 96 samples mix at least 2880 ul of NucleoMag Plant 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 Plant Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has formed 14 MACHEREY NAGEL 05 2005 Rev 01 NucleoMag 96 Plant 5 Separate the magnetic beads against the side of the wells by placing the Square well Block on the magnetic separator Wait at least 2 min until all the beads have been attracted t
3. the beads are completely resuspended Shake the storage bottle well or place it on a vortex 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 on each system It is recommended to use 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 pipetting up and down is more efficient than mixing by a shaker or magnetic mix Method Resuspension Speed Small elution Number of tips B volume Magnetic mix mix ot a famed 8 channel pipetting device 2 5 Elution procedures Purified genomic DNA can be eluted directly with t
4. Genomic DNA from Plant User manual NucleoMag 96 Plant May 2005 Rev 01 MACHEREY NAGEL ah Genomic DNA from Plant Table of contents 1 Kit contents 2 Product description 2 1 The basic principle 2 2 Magnetic separation systems 2 3 Adjusting the shaker settings 2 4 Handling of beads 2 5 Elution procedures 2 6 Storage and homogenization of samples 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 General procedure 5 1 Standard protocol for the purification of genomic DNA 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty MACHEREY NAGEL 05 2005 Rev 01 O O WANN DDO OT OH RA Se Con N KR gt De E E O O N Genomic DNA from Plant 1 Kit contents 1 x 96 preps Cat No 744 400 1 NucleoMag C Beads 3 ml Lysis Buffer MC1 60 ml Binding Buffer MC2 2x25 ml Wash Buffer MC3 75 ml Wash Buffer MC4 75 ml Wash Buffer MCS 75 ml Elution Buffer MC6 25 ml RNase A 15 mg Elution plate U bottom including one Self 1 adhering PE foil Protocol 1 Material to be supplied by user 80 Ethanol Separation plate e g Square well Block Homogenization tubes e g MN Tube Strips and Cap Strips see ordering information section 6 2 4 MACHEREY NAGEL 05 2005 Rev 01 NucleoMag 96 Plant 4 x 96 preps 744 400 4 12 ml 240 ml 8 x 25 ml 2 x150 ml 2 x 150 ml 2 x150 ml 100 ml 2 X 30
5. ant protocol prepare the following e RNase A Add the indicated volume of water to RNase A Store at 4 C e 80 Ethanol use molecular biology grade ethanol dilute with appropriate water to 80 NucleoMag 96 Plant 1 x 96 preps 4 x 96 preps 24 x 96 preps Cat No 744400 1 744400 4 744400 24 1x 15 mg 2x 30 mg 12 x 30 mg RNase A add 1 25 ml water add 2 5 ml water to add 2 5 ml water lyophilized each vial to each vial MACHEREY NAGEL 05 2005 Rev 01 9 NucleoMag 96 Plant 4 Safety instructions risk and safety phrases 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 Component Hazard Hazard Risk Safety Contents Symbol Phrases Phrases isopropanol Highly flammable Irritating to eyes R 11 36 67 S 7 16 MC2 Vapours may cause drowsiness and 24 25 26 dizziness w Xi sodium 4 Xn Flammable Harmful if swallowed R 10 22 S 7 16 MC3 perchlorate in Irritating to eyes and skin 36 38 ethanol sodium 4 a Harmful if swallowed Flammable R 22 10 S 13 22 27 MC4 perchlorate in ethanol RNase A 4 Xj May cause sensitization by inhalation R 42 43 S 7 16 22 RNase A lyophilized and skin contact Risk Phrases R 10 Flammable R 11 Highly flammable R 22 Harmful if swallowed R 36 Irritating to eyes R 36 38 Irritating to eyes and skin R 42 43 May cause sensitization by inhalation and ski
6. he supplied Elution Buffer MC6 Elution can be carried out in a volume of 2 50 ul It is essential to cover the NucleoMag Plant Beads completely with elution buffer during the elution step The volume of dispensed elution buffer depends on the magnetic separation system 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 high 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 05 2005 Rev 01 1 Genomic DNA from Plant 2 6 Storage and homogenization of samples We recommend to use young plant samples and if possible to keep plants for about 12 h in 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 commerc
7. ial 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 authorized 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
8. ial 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 Leopoldshohe Tissue Striker www KisanBiotech com or Geno Grinder 2000 www spexcsp com or for Germany www c3 analysentechnik de Homogenizing 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 Gottingen 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 8 MACHEREY NAGEL 05 2005 Rev 01 Genomic DNA from Plant 3 Storage conditions and preparation of working solutions Attention Buffers MC3 and MC4 contain chaotropic salt Wear gloves and goggles Buffer MC2 is highly flammable and irritant All components of the NucleoMag 96 Plant kit should be stored at room temperature 20 25 C and are stable for up to one year All buffers are delivered ready to use Before starting any NucleoMag 96 Pl
9. is colorless magnetic bead pellet is clearly visible 9 Add 600 ul Wash Buffer MC5 to each well and incubate for 60 s while the beads are still separated on the magnet Then aspirate and discard the supernatant Note Do not resuspend the beads in Wash Buffer MC 5 This step is to remove traces of ethanol and eliminates a drying step MACHEREY NAGEL 05 2005 Rev 01 15 NucleoMag 96 Plant 10 Add desired volume of Elution Buffer MC6 50 200 ul to each well and resuspend 16 the bead DNA complex by shaking 5 10 min Alternatively pipette up and down 10 times and incubate 5 10 min Separate the magnetic beads against the side of the wells by placing the Square well Block on the magnetic separator Wait 2 min until all the beads have been attracted to the magnet Transfer the supernatant containing the purified genomic DNA to the Elution Plate Note The yield can be increased by 15 20 by using prewarmed elution buffer 55 C or by incubating the bead elution buffer suspension at 55 C for 10 min MACHEREY NAGEL 05 2005 Rev 01 Genomic DNA from Plant 6 Appendix 6 1 Troubleshooting Problem Poor DNA yield Low purity Possible cause and suggestions Elution buffer volume insufficient e Beads pellet must be covered completely with elution buffer Insufficient performance of elution buffer during elution step e Remove residual buffers during the separation steps completely Remaining buffers dec
10. magnetic systems Separators with moving magnetic pins e g Te MagS for automated use only 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 Separation takes place when the system stops 2 3 Adjusting the shaker settings When using a plate shaker for the washing and elution steps the speed settings have to be checked carefully for each specific separation plate and shaker to prevent cross contamination from well to well Proceed as follows e Apply 600 ul dyed water select desired elution buffer volume to the wells of the separation plate Position the plate on the shaker and start shaking with a moderate speed setting for 30 seconds Turn off the shaker and check plate surface for small droplets of dyed water e Increase speed setting shake for an additional 30 seconds and check plate surface for droplets again e 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 and elution step 6 MACHEREY NAGEL 05 2005 Rev 01 Genomic DNA from Plant 2 4 Handling of beads Distribution of beads A homogenous 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
11. mg 24 x 96 preps 744 400 24 72 ml 3 x 480 ml 3 x 400 ml 2 x 900 ml 2 x 900 ml 2 x 900 ml 600 ml 12 x 30 mg 24 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 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 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 s DNA A Contaminants g NucleoMag Beads H ae Plant tissue is extracted with Lysis Buffer MC1 f 7T Binding conditions are adjusted and the NucleoMag Pa Plant Beads are added to the sample A C at ii i DNA is bound to the NucleoMag Plant Beads Beads are held back in the well while contaminants are ll L si washed away ra j he j EN af _i j j j a W m DNA is eluted from the beads and reco
12. n RT co optional mix by pipetting up and down 2 min separation 1 oe T re g t i FA S Ti intel 7 80 Ethanol wash step 600 ul 80 Ethanol shake 5 min RT co optional mix by pipetting up and down 2 mi ti I min separation HN pinl 8 MC5 wash step 600 ul MC5 60 sec incubation aspirate and discard supernatant re Note Do not resuspend beads in B MC5 buffer leer 12 MACHEREY NAGEL 05 2005 Rev 01 NucleoMag 96 Plant 9 Elute genomic DNA and transfer to Elution Plate 50 200 ul MC6 shake 5 min RT 2 min separation transfer Optional Elution at 55 C MACHEREY NAGEL 05 2005 Rev 01 co Pe se ee S dJ tye a EE 13 NucleoMag 96 Plant 5 1 Standard protocol for the purification of genomic DNA This protocol is designed for magnetic separators with static pins e g NucleoMag SEP and suitable plate shakers e g H P Variomag Teleshake This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments For the availability of ready to run scripts please contact your local distributor or MN directly 1 Homogenize and lyse sample material Homogenize about 20 50 mg lyophilized plant tissue e g using mictrotube strips in a mixer mill and add 500 ul MC1 lysis buffer Do not moisten the rim Close the individual wells with cap strips Mix by vigorous shaking for 15 30 sec Spin briefly for 30 sec at 1 500 x g to
13. n contact R 67 Vapours may cause drowsiness and dizziness Safety Phrases S7 Keep container tightly closed S 13 Keep away from food drink and animal feedingstuffs S 16 Keep away from sources of ignition No smoking S 22 Do not breathe dust S 24 Avoid contact with skin S 24 25 Avoid contact with skin and eyes S 27 Take off immediately all contaminated clothing Label not necessary if quantity below 25 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 10 MACHEREY NAGEL 05 2005 Rev 01 NucleoMag 96 Plant 5 General procedure Homogenize and lyse plant samples Homogenization 20 50 mg add 500 ul MC buffer mix and incubate at 56 C 30 min Clear lysate by centrifugation transfer 400 ul of cleared lysate to a Square 5 600 xg well Block for further processing Bind DNA to NucleoMag Plant Beads 400 ul cleared lysate 30 ul C beads 400 ul MC2 shake 5 min at RT Remove supernatant 2 min separation MC3 wash step 600 ul MC3 shake 5 min RT optional mix by pipetting up and down 2 min separation MACHEREY NAGEL 05 2005 Rev 01 AE o j H h SSX 0 issn a at co I TAS d A J ve J J 11 NucleoMag 96 Plant 6 MC4 wash step 600 ul MC4 shake 5 mi
14. o the magnet Remove and discard supernatant by pipetting Remove the Square well Block from the magnetic separator 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 6 Add 600 ul Wash Buffer MC3 to each well and wash the bead DNA complex by shaking 5 min at room temperature Alternatively pipette up and down 15 times Separate all of the magnetic beads against the side of the well by placing the Square well Block on the magnetic separator as described above Aspirate and discard the supernatant Remove the Square well Block from the magnetic separator 7 Add 600 ul Wash Buffer MC4 to each well and wash the bead DNA complex by shaking 5 min at room temperature Alternatively pipette up and down 15 times Separate all of the magnetic beads against the side of the well by placing the Square well Block on the magnetic separator as described above Aspirate and discard the supernatant 8 Add 600 ul 80 ethanol to each well and wash the bead DNA complex by shaking 5 min at room temperature Alternatively pipette up and down 15 times Separate all of the magnetic beads against the side of the well by placing the Square well Block on the magnetic separator as described above Aspirate and discard the supernatant Do not remove the Square well Block from the magnetic separator Note Supernatant
15. of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 and 275 e mail TECH BlIO mn net com 20 MACHEREY NAGEL 05 2005 Rev 01
16. rease efficiency of following wash steps and elution step Beads dried out e Do not let the beads dry as this might result in lower elution efficiencies Partial elution in Wash Buffer MC5 already e 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 e 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 e Mix immediately after dispensing NucleoMag Plant Beads Binding Buffer MC2 to the lysate Insufficient washing procedure e Use only the appropriate combinations of separator and plate e g MN Square well Block in combination with NucleoMag SEP MACHEREY NAGEL 05 2005 Rev 01 17 Problem Suboptimal performance of DNA in downstream applications Carry over of beads Cross contamination 18 Genomic DNA from Plant Possible cause and suggestions Carry over of ethanol from 80 ethanol wash solution e Be sure to remove all of the ethanolic wash solution as residual ethanol interferes with downstream applications Low purity e see above Time for magnetic separation too short e Increase separation time to allow the beads to be completely attracted to the magnetic pins before
17. 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 an extra copy MACHEREY NAGEL does not warrant against 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 against defects in products or components not manufactured by MACHEREY NAGEL or against 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 MACHEREY NAGEL 05 2005 Rev 01 19 Genomic DNA from Plant 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 spec
18. vered while beads are held back in the well by the magnet DNA is ready to use in downstream applications MACHEREY NAGEL 05 2005 Rev 01 Genomic DNA from Plant 2 2 Magnetic separation systems For use of NucleoMag 96 Plant the use of the magnetic separator NucleoMag SEP is recommended Separation is carried out in an MN Square well Block see ordering information The kit can also be used with other common separators See suppliers ordering information for suitable separation plates Magnetic separator Separation plate or tube ea te ee Square well Block MN Cat No 740 670 Promega MagnaBot Square well Block MN Cat No 740 670 Tecan Te MagS 1 5 ml tubes without lid Sarstedt Static magnetic pins Separators with static magnetic pins e g NucleoMag SEP for manual use and for use on liquid handling workstations This type of separator is recommended in combination with a plate shaker e g H P Variomag Teleshake H P Labortechnik AG Bruckmannring 28 D 85764 Oberschleigheim Germany www hp lab de 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
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