NucleoMag® Blood 200 µL - MACHEREY
Contents
1. Genomic DNA from blood User manual NucleoMag Blood 200 uL May 2014 Rev 03 MACHEREY NAGEL www mn net com Genomic DNA from blood Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Magnetic separation systems 7 2 4 Adjusting the shaker settings 8 2 5 Handling of beads 8 2 6 Elution procedures 9 3 Storage conditions and preparation of working solutions 11 4 Safety instructions 12 5 Protocol for the isolation of DNA from blood 14 6 Appendix 20 6 1 Troubleshooting 20 6 2 Ordering information 22 6 3 Product use restriction warranty 22 MACHEREY NAGEL 05 2014 Rev 03 3 Genomic DNA from blood 1 Components 1 1 Kit contents NucleoMag Blood 200 uL 1x 96 preps 4 x 96 preps REF 744501 1 744501 4 NucleoMag B Beads 3 mL 12 mL Lysis Buffer MBL1 13 mL 45 mL Binding Buffer MBL2 40 mL 160 mL Wash Buffer MBL3 300 mL 900 mL Wash Buffer MBL4 125 mL 500 mL Elution Buffer MBL5 30 mL 125 mL Proteinase K lyophilized 50 mg 4x50 mg Proteinase Buffer PB 8 mL 15 mL User manual 1 1 Elution Buffer MBL5 5 mM Tris pH 8 5 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood 1 2 Reagents consumables and equipment to be supplied by user Reagents 802. noO RT DN O 50 75 100 125 150 175 Elution volume uL Figure 1 Influence of elution volume on DNA yield example MACHEREY NAGEL 05 2014 Rev 03 9 Genomic DNA from blood Elution is possible at room temperature However DNA yield can be increased by 15 20 if elution is performed at 72 C see Figure 2 8 7 0 T 6 45 24 33 2 1 0 RT 56 C 72 C Figure 2 Influence of elution temperature on DNA yield 10 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood 3 Storage conditions and preparation of working solutions Attention Buffers MBL1 MBL2 and MBL3 contain chaotropic salt Wear gloves and goggles CAUTION Buffer MBL1 contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All components of the NucleoMag Blood 200 uL 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 NucleoMag Blood 200 uL protocol prepare the following Add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K see table below Proteinase K solution is stable at 20 C for up to 6 months NucleoMag Blood 200 uL 1x 96 preps 4 x 96 preps REF 744501 1 744501 4 Proteinase K 50 mg 4x 50 mg Add 2 5 mL Add 2
3. or operation 24 MACHEREY NAGEL 05 2014 Rev 03 MACHEREY NAGEL MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Germany Switzerland France USA and international MACHEREY NAGEL AG MACHEREY NAGEL EURL MACHEREY NAGEL Inc Tel 49 24 21 969 0 Tel 41 62 388 55 00 Tel 33 388 68 22 68 Tel 1 484 821 0984 E mail info mn net com E mail sales ch mn net com E mail sales fr mn net com E mail sales us mn net com A039476 0350 1
4. 5 mL Proteinase Buffer PB Proteinase Buffer PB to each vial MACHEREY NAGEL 05 2014 Rev 03 11 Genomic DNA from blood 4 Safety instructions The following components of the NucleoMag Blood 200 uL kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g 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 MBL1 Guanidine hydrochloride Danger 302 315 280 301 312 50 66 319 302 352 Guanidinhydrochlorid 50 66 Gefahr 305 351 338 330 332 313 337 313 MBL2 Sodium perchlorate 20 Warning 226 302 210 233 40 ethanol 35 55 301 312 330 Natriumperchlorat 20 40 Achtung 403 235 Ethanol 35 55 MBL3 Sodium perchlorate amp Warning 226 210 233 5 20 ethanol 20 35 403 235 Natriumperchlorat 5 20 Achtung Ethanol 20 35 Proteinase K Proteinase K lyophilized Danger 315 319 261 280 Proteinase K lyophilisiert D Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 342 311 403 233 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmfu
5. already yield Keep the separation plate on the magnet while dispensing Wash Buffer MBL4 Do not resuspend beads in this buffer and do not incubate beads in this buffer for more than 2 min as Buffer MBL4 promotes DNA elution 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 Mix immediately after dispensing NucleoMag B Beads and Binding Buffer MBL2 to the lysate Poor blood quality Be sure that no blood clots are transferred to the well Blood can be stored at 2 8 C for two weeks Freeze samples if stored for longer periods Low purity Insufficient washing procedure Use only the appropriate combinations of separator and plate for example Square well Block in combination with NucleoMag SEP 20 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood Problem Possible cause and suggestions Suboptimal Carry over of ethanol from ethanol wash step performance Be sure to remove all of the ethanol from the ethanol wash of DNAin step Carry over of ethanol may interfere with downstream downstream applications Use of Buffer MBL4 or introduce on air drying step applications Low purity See above Time for magnetic separation too short Increase separation time to allow the beads to be completely attracted to the magnetic pins before aspirating any liquid
6. and discard the 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 MBL3 1 wash Remove the Square well Block from the magnetic separator Add 800 uL Buffer MBL3 to each well and resuspend the bead DNA complex by shaking at room temperature until the beads are resuspended completely 5 min Alternatively resuspend the beads by pipetting up and down 15 times Note Make sure that the magnetic beads are resuspended completely and form a brownish suspension If necessary increase shaking incubation time or number of mixing cycles Incomplete mixing may result in low purity of eluted DNA Separate the magnetic beads by placing the Square well Block on the magnetic separator Wait for at least 2 min until all the beads have been attrected to the magnet Remove and discard supernatant by pipetting Note Supernatant has a brownish color magnetic bead pellet is now visible Wash with MBL3 2 wash Remove the Square well Block from the magnetic separator Add 800 pL Buffer MBL3 to each well for a second wash step with Buffer MBL3 Wash the bead DNA complex by shaking 5 min at room temperature Alternatively resuspend the beads by pipetting up and down 15 times Separate the magnetic beads by placing the Square well Block on the magnetic separator Wait for
7. at least 2 min until all the beads have been attrected to the magnet Remove and discard supernatant by pipetting Note Supernatant is colorless magnetic bead pellet is clearly visible MACHEREY NAGEL 05 2014 Rev 03 NucleoMag Blood 200 uL Wash with 80 ethanol Remove the Square well Block from the magnetic separator Add 800 uL 80 ethanol to each well and wash the bead DNA complex by shaking 5 min at room temperature Alternatively resuspend the beads by pipetting up and down 15 times Separate the magnetic beads by placing the Square well Block on the magnetic separator Wait for at least 2 min until all the beads have been attrected to the magnet Remove and discard supernatant by pipetting Note Supernatant is colorless magnetic bead pellet is visible now Wash with MBL4 Leave Square well Block on the magnetic separator Gently add 900 uL Buffer MBL4 to each well and incubate for 45 90 s while the beads are still attracted to the magnet Then aspirate and discard the supernatant Note Do not resuspend the beads in Buffer MBL4 This step is to remove traces of ethanol and eliminates a drying step Optional Washing the magnetic beads with Buffer MBL4 may decrease the DNA yield slightly Alternatively replace this washing step by air drying of the magnetic beads for 10 15 min until all of the ethanol from previous washing step has evaporated Beads with remaining ethanol appear to be
8. ethanol for the washing step Equipment Consumables Product REF Pack of 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 Elution plate for collecting purified DNA e g Elution Plate U bottom 96 well 0 3 mL 740486 24 24 microtiterplate with 300 uL U bottom wells For use of kit on KingFisher 96 and KingFisher Flex instrument KingFisher 96 Accessory Kit B Square 740951 well Blocks Deep well tip Combs Elution Plates for 4 x 96 NucleoMag 96 Blood 200 uL preps using KingFisher 96 platform 1 set MACHEREY NAGEL 05 2014 Rev 03 5 Genomic DNA from blood 2 Product description 2 1 The basic principle The NucleoMag Blood 200 uL procedure is based on reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions Whole blood is lysed with Lysis Buffer MBL1 and Proteinase K Following lysis incubation magnetic beads are added and binding conditions under which the DNA binds to the magnetic beads are adjusted by addition of Binding Buffer MBL2 After magnetic separation and removal of the supernatant the paramagnetic beads are washed three times to remove contaminants and salt There is no need for a drying step as ethanol from previous wash steps is removed by Wash Buffer MBL4 Finally highly purified DNA is eluted with low salt Elution Buffer MBL5 and can directly be used fo
9. from the well Carry over sce Aspiration speed too high elution ste of beads p E gn p High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step To remove magnetic beads from the eluates put the elution plate on the magnetic separator and aspirate the supernatant after sufficient beads separation Cross conta mination Contamination of the rims Do not moisten the rims of the Square well Block when transferring the blood 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 2014 Rev 03 21 Genomic DNA from blood 6 2 Ordering information Product REF Pack of NucleoMag Blood 200 uL 744501 1 1 x 96 preps 744501 4 4x 96 preps NucleoMag SEP 744900 1 Square well Blocks 740481 4 4 740481 24 24 Elution Plates U bottom 740486 24 24 Self adhering PE Foil 740676 50 sheets KingFisher 96 Accessory Kit B 744951 1 set Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag Blood 200 uL preps using KingFisher 96 platform Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoMag Blood 200 uL kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly desc
10. glossy Moderate heating 37 C can support and shorten the air drying step Over drying the beads may result in low yield in the final elution step Elute DNA Remove the Square well Block from the magnetic separator Add desired volume of Buffer MBL5 50 100 uL to each well of the Square well Block and resuspend the bead DNA complex by shaking 5 10 min Alternatively resuspend the beads by pipetting up and down 15 times Separate the magnetic beads by placing the Square well Block on the magnetic separator Wait for at least 2 min until all the beads have been attrected to the magnet Transfer the supernatant containing the purified genomic DNA to the Elution Plate Note Yield can be increased by 15 20 by using pre heated elution buffer 55 72 C or by incubating the bead elution buffer suspension at 55 72 C for 10 min MACHEREY NAGEL 05 2014 Rev 03 19 Genomic DNA from blood 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions 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 completely Remaining buffers decrease efficiency of subsequent wash steps and elution step Beads dried out Do not let the beads dry as this might result in lower elution efficiencies Poor DNA Partial elution in Wash Buffer MBL4
11. 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
12. 200 uL NucleoMag Blood 200 pL can be processed completely at room temperature however elution at 55 C or 72 C will increase the yield by about 15 20 NucleoMag Blood Beads are highly reactive superparamagnetic beads The binding capacity is approximately 0 4 ug of gDNA per 1 uL of NucleoMag Blood Bead Suspension 1 uL of suspension contains 140 ug of beads 6 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood 2 3 Magnetic separation systems For use of NucleoMag Blood 200 uL 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 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 tra
13. ate 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 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 MBL2 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 the 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 to use the separation plates or tubes specified by the su
14. athing BEI EINATMEN 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 sp len Call a POISON CENTER doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len IF skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung rztliche Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep cool Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren 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 05 2014 Rev 03 13 NucleoMag Blood 200 uL 5 Protocol for the isolation of DNA from blood Protocol at a glance Fo
15. 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 components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components
16. g SEP 900 pL MBL4 Incubate for 45 90 s Asprirate and discard supernatant Note Do not resuspend the beads in Buffer MBL4 Elute DNA Remove Square well Block from NucleoMag SEP 50 100 pL MBL5 Optional Elute at 55 C Shake 5 10 min at RT Optional Mix by pipetting up and down Separate 2 min and transfer DNA into elution plate tubes 16 MACHEREY NAGEL 05 2014 Rev 03 NucleoMag Blood 200 uL Detailed protocol This protocol is designed for magnetic separators with static 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 Proteinase K was prepared according to section 3 1 Lyse samples Dispense 20 uL of Proteinase K solution into each well of a Square well Block Transfer 200 uL blood equilibrated to room temperature to each well of 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 Add 80 uL Buffer MBL1 to each sample and mix by repeated pipetting up and down 3 5 times and shaking for 5 10 min at room temperatu
17. l if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizugen H 319 Causes serious eye irritation Verursacht schwere Augenreizung 12 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood Hazard phrases H 334 H 335 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 210 P 233 P 261 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 312 P 330 P 332 313 P 337 313 P 342 311 P 403 233 P 403 235 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten 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 SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for bre
18. nsferred to the shaker module for resuspension of the beads Movable magnetic systems Separators with moving magnetic pins for example 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 the movement Automated separators Separators with moving magnets for example Thermo Fisher Scientific KingFisher instruments 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 MACHEREY NAGEL 05 2014 Rev 03 7 Genomic DNA from blood 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 wash steps Load 800 uL 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 plate surface for small droplets of dyed water Increase speed setting shake for an additional 30 seconds and check pl
19. 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 MACHEREY NAGEL 05 2014 Rev 03 23 Genomic DNA from blood 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
20. pplier of the magnetic separator 8 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood 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 however is more efficient than mixing by a shaker or magnetic mix Complete and homogenous resuspension of the beads in wash buffers MBL3 MBL4 and 80 ethanol is mandatory for best performance of the kit Method Resuspension Speed Number of tips efficiency needed Magnetic mix Shaker Low Pipetting acceptable good excellent 2 6 Elution procedures Purified total DNA can be eluted directly with the supplied Elution Buffer MBL5 Elution can be carried out in a volume of gt 50 uL It is essential to cover the NucleoMag B Beads completely with Elution Buffer MBL5 during the elution step The volume of dispensed Elution Buffer MBL5 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 MBL5 For some separators high elution volumes might be necessary to cover the whole magnetic bead pellet ug DNA o
21. r additional equipment and hardware requirements refer to section 1 2 and 2 3 respectively For detailed information on each step see page 19 Before starting the preparation Check if Proteinase K was prepared according to section 3 1 Lyse samples Dispense 20 uL Proteinase K into Square well Block 200 uL blood 80 pL MBL1 Mix 3 5 times Shake 10 min at RT 2 Bind DNA to 25 uL B Beads NucleoMag B Beads 300 pL MBL2 Mix by shaking for 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 3 Wash with MBL3 Remove Square well Block 1 wash from NucleoMag SEP 800 pL MBL3 14 MACHEREY NAGEL 05 2014 Rev 03 NucleoMag Blood 200 uL Resuspend Shake 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation Wash with MBL3 Remove Square well Block 2 wash from NucleoMag SEP 800 pL MBL3 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 800 uL 80 ethanol Resuspend Shake 5 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation MACHEREY NAGEL 05 2014 Rev 03 15 NucleoMag Blood 200 uL Wash with MBL4 Leave Square well Block on NucleoMa
22. r downstream applications The NucleoMag Blood 200 uL kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators 2 2 Kit specifications NucleoMag Blood 200 uL is designed for rapid manual and automated small scale preparation of highly pure genomic DNA from 200 uL whole blood using the NucleoMag 96 SEP 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 obtained DNA can be used directly as template for PCR blotting or any kind of enzymatic reactions NucleoMag Blood 200 pL allows easy automation on common liquid handling instruments or automated magnetic separators for example Thermo Scientific s KingFisher instruments The actual processing time depends on the configuration of your instrument and the magnetic separation system used Typically 96 samples can be purified in less than 120 minutes using the NucleoMag SEP on the automation platform The kit provides reagents for the purification of 2 8 ug of pure genomic DNA from 200 uL whole blood with an A A ratio 1 6 1 9 and typical concentration of 20 40 ng L Depending on the health status of the blood donor and the elution volume used concentrations of 10 160 ng uL can be obtained Fresh or frozen blood treated either with EDTA or citrate can be used The procedure is optimized for a sample volume of
23. re Alternatively when processing the kit without a shaker pipette up and down 10 times and incubate 5 10 min at room temperature 2 Bind DNA to NucleoMag B Beads Add 25 uL NucleoMag B Beads to each sample Mix magnetic beads thoroughly before dispensing to the samples Add 300 uL Buffer MBL2 to each sample and mix by pipetting up and down 3 5 times and shake for 5 min to allow the DNA to bind to the magnetic beads Alternatively when processing the kit without a shaker pipette up and down 10 times and incubate 5 min at room temperature Note NucleoMag B Beads and Buffer MBL2 can be premixed For each sample to be processed mix 25 uL of NucleoMag B Beads with 300 uL Buffer MBL2 Vortex briefly Depending on the dead volume of the reservoir additional amounts of bead suspension and binding buffer are necessary Mix the solution several times to avoid the beads to settle within the premix distribution step Do not store the premix of the NucleoMag B Beads and Buffer MBL2 longer than 12 h Be sure to resuspend the NucleoMag B Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has been formed MACHEREY NAGEL 05 2014 Rev 03 17 NucleoMag Blood 200 uL 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 by the magnet Remove
24. ribed 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 N VITRO USES ONLY 22 MACHEREY NAGEL 05 2014 Rev 03 Genomic DNA from blood 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 expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN 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 or
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