Genomic DNA from forensic samples User manual
Contents
1. Genomic DNA from forensic samples User manual NucleoMag Forensic August 2013 Rev 01 MACHEREY NAGEL MN www mn net com Viral RNA DNA isolation Table of contents 1 Components 1 1 Kit contents 1 2 Material to be supplied by user Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Magnetic separation systems 2 4 Adjusting the shaker settings 2 5 Handling of beads 2 6 Handling of Proteinase K 2 7 Elution procedures Storage conditions and preparation of working solutions Safety instructions 4 1 Risk and safety phrases 4 2 GHS classification Protocol 5 1 Sample material 5 2 Isolation of genomic DNA from forensic samples Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty 61 oo oo ON 9 09 9 DD 10 11 11 12 14 14 14 19 19 20 21 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 1 Components 1 1 Kit contents NucleoMag Forensic 1x 96 preps 4 x 96 preps REF 744650 1 744650 4 NucleoMag F Beads 1 4mL 4x 1 4 mL Lysis Buffer FEB 50 mL 4x 50 mL Binding Buffer FBB 90 mL 4x 90 mL Wash Buffer FWB1 70 mL 4x 70 mL Wash Buffer FWB2 Concentrate 50 mL 4x 50 mL Elution Buffer FEL 30 mL 2x 30 mL Proteinase K lyophilized 3 x 40 mg 6x75 mg Proteinase Buffer PB 8 mL 35 mL User manual 1 1 For preparation of working solutions and storage conditions see section 3 4 MA2. 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 Hazard Precaution symbol phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze FBB Sodium perchlorate 20 Danger 226 302 210 233 301 312 40 ethanol 35 55 330 403 235 Natriumperchlorat 20 40 Gefahr Ethanol 35 55 FWB1 Sodium perchlorate Warning 226 210 233 403 235 5 20 ethanol 20 35 Natriumperchlorat 5 20 Achtung Ethanol 20 35 Proteinase K Proteinase K lyophilized m Danger 315 317 261 271 280 Proteinase K Iyophilisiert Gefahr 319 334 302 352 304 340 335 305 351 338 312 D 333 313 337 313 342 311 362 403 233 405 Hazard phrases H 226 H 302 H 315 H 317 H 319 H 334 H 335 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar Harmful if swallowed Gesundheitssch dlich bei Verschlucken Causes skin irritation Verursacht Hautreizungen 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 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 Keep away from heat sp
3. 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 or operation 22 MACHEREY NAGEL 08 2013 Rev 01
4. 50 sheets KingFisher 96 Accessory Kit A 744950 1 set Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag Forensic preps using KingFisher 96 platform Visit www mn net com for more detailed product information 20 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 6 3 Product use restriction warranty NucleoMag Forensic kit components are intended developed designed and sold FOR RESEARCH PURPOSES 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 N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO
5. 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 08 2013 Rev 01 21 DNA isolation from forensic samples components not manufactured by MACHEREY 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 an
6. fluids Additionally forensic samples such as cigarette butts or chewing gum can be used as starting material Examples of appropriate sample types and inputs are listed in the table below However each lab should perform studies to independently validate input amounts It is important that the sample is covered with Iysis buffer during Iysis procedure sample amounts might have to be adapted Sample type Example sample input Saliva on swabs Up to 50 uL swab one swab Blood Blood on swabs Up to 5 uL undiluted Blood FTA paper or fabric Up to 8 mm diameter Body fluids on fabric Up to 8 mm diameter Body fluids on swabs Up to one swab Chewing gum Up to one chewing gum Cigarette butt Up to one butt 5 2 Isolation of genomic DNA from forensic samples Protocol at a glance e For hardware requirements refer to section 2 3 For detailed information on each step see page 17 Before starting the preparation Check that Proteinase K was prepared according to section 3 14 MACHEREY NAGEL 08 2013 Rev 01 NucleoMag Forensic Lyse sample To each sample add 450 pL FEB 45 uL Proteinase K 5 uL 1M DTT Mix 56 C 1 h or overnight Bind DNA to 800 pL FBB NucleoMag F Beads 12 uL F Beads Mix by shaking for 10 min at RT Optional Mix by pipetting up and down Remove supernatant after 2 min separation 3 Wash with FWB1 Remove Square well Block from NucleoMag S
7. the wash and elution steps Separation 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 600 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 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 08 2013 Rev 01 7 DNA isolation from forensic samples 2 5 Handling of bea
8. under appropriate buffer conditions Sample lysis is achieved by incubation with a Lysis Buffer FEB containing chaotropic ions supported by Proteinase K digestion For binding of nucleic acids to the paramagnetic beads Binding Buffer FBB and the NucleoMag F Beads are added to the lysate After magnetic separation the paramagnetic beads are washed to remove contaminants and salts using Wash Buffers FWB1 and FWB2 Residual ethanol from previous wash steps is removed by airdrying Finally highly pure DNA is eluted with low salt Elution Buffer FEL Purified DNA can directly be used for downstream applications e g STR analysis The NucleoMag Forensic kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators 2 2 Kit specifications NucleoMag Forensic is designed for rapid manual and automated small scale preparation of DNA from swabs The kit is designed for use with NucleoMag SEP magnetic separator 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 or STR analysis NucleoMag Forensic 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 purif
9. 6 Handling of Proteinase K For dispensing the Proteinase K solution to each sample it is recommended to predispense the needed amount to a separate reaction tube Using a liquid handling device we recommend to dispense the needed Proteinase K solution 45 uL per prep with 10 extra volume in a suitable tube for the correspondent robot Unused Proteinase K solution should be stored at 20 C for further extractions 8 channel pipetting device 8 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 2 7 Elution procedures Purified DNA can be eluted directly with the supplied Elution Buffer FEL Elution can be carried out in a volume of 25 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 high elution volumes might be necessary to cover the whole pellet MACHEREY NAGEL 08 2013 Rev 01 9 DNA isolation from forensic samples 3 Storage conditions and preparation of working solutions Attention Buffers FEB FBB and FWB1 contain chaotropic salt Wear gloves and goggles All components of the NucleoMag Forensic kit should be stored at room temperature 18 25 C and are
10. CHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 1 2 Material to be supplied by user Product REF Pack of Separation plate for magnetic beads separation e g Square well Block 96 well block 740481EO 4 with 2 1 mL square wells ethylene oxide treated Lysis tubes for incubation of samples and lysis u l 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 3mL microtiterplate with 300 uL flat bottom wells 740673 20 For use of kit on KingFisher 96 instrument e g KingFisher 96 Accessory Kit A Square well Blocks Deep well tip combs 744950 1 set Elution Plates for 4 x 96 NucleoMag Forensic preps using KingFisher 96 platform Reagents 96 100 ethanol e 1MDTT solution MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 2 Product description 2 1 The basic principle The NucleoMag Forensic kit is designed for the isolation of DNA from swabs derived from forensic casework samples This kit provides reagents and magnetic beads for isolation of 96 or 384 samples The procedure is based on the reversible adsorption of nucleic acids to paramagnetic beads
11. EP 600 pL FWB1 Resuspend Shake 1 min at RT Remove supernatant after 2 min separation 4 Wash with FWB2 1 Remove Square well Block from NucleoMag SEP 600 pL FWB2 Resuspend Shake 1 min at RT MACHEREY NAGEL 08 2013 Rev 01 15 NucleoMag Forensic Remove supernatant after 2 min separation 5 Wash with FWB2 2 9 Remove Square well Block from NucleoMag SEP 600 pL FWB2 Resuspend Shake 1 min at RT Remove supernatant after 2 min separation 6 Air dry magnetic beads Air dry 15 min at RT 7 Elute DNA Remove Square well Block from NucleoMag SEP 25 50 pL FEL Shake 10 min at 70 C Optional Mix by pipetting up and down Separate 2 min and transfer DNA into elution plate tubes 16 MACHEREY NAGEL 08 2013 Rev 01 NucleoMag Forensic Detailed protocol This protocol is designed for magnetic separators with static pins e g NucleoMag SEP and suitable plate shakers It is recommended using a Square well Block for separation see ordering information 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 1 Lyse sample Add 45 uL Proteinase K 5 uL 1M DTT and 450 uL Lysis Buffer FEB to a reaction tube containing the sample Mix well by repeated pipetting up and dow
12. N CENTER or doctor physician if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len If skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche 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 Take off contaminated clothing Kontaminierte Kleidung ausziehen Store in a well ventilated place 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 Store locked up Unter Verschluss 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 A The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin MACHEREY NAGEL 08 2013 Rev 01 13 NucleoMag Forensic 5 Protocol 5 1 Sample material The NucleoMag Forensic kit can be used to isolate DNA from most forensic sample types including body fluids stains and swabs of body
13. arks 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 12 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples Precaution phrases P 271 P 280 P 301 312 P 302 352 P 304 340 P 305 351 388 P 312 P 330 P 333 313 P 342 311 P 337 313 P 362 P 403 233 P 403 235 P 405 Use only outdoors or in a well ventilated area Nur im Freien oder in gut bel fteten R umen verwenden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell Bei Verschlucken Bei Unwohlsein Giftinformationszentrum oder Arzt anrufen IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen IF INHALED Remove to fresh air and keep at rest in a position comfortable for beathing Bei Einatmen Die betroffene Person an die frische Luft bringen und ftir ungehinderte Atmung sorgen 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 Call a POSIO
14. d 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 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
15. 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 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 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
16. ds 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 During automation a premix step before aspirating the beads suspension 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 pipetting 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 2
17. ied in less than 120 minutes using the NucleoMag SEP on the automation platform 2 3 Magnetic separation systems For use of NucleoMag Forensic 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 740481EO Tecan Te MagS 1 5 mL tubes without lid Sarstedt 6 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 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
18. leoMag Forensic 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 FWB2 1 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL Buffer FWB2 and resuspend the beads by shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down 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 FWB2 2 9 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL Buffer FWB2 and resuspend the beads by shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down 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 Air dry magnetic beads Air dry the magnetic bead pellet for 15 min at room temperature Elute DNA Add desired volume of Buffer FEL 25 50 uL to each well of the Square well Bl
19. lyophilisiert 42 26 36 37 Risk phrases R10 Flammable Entz ndlich R 22 Harmful by inhalation Gesundheitssch dlich beim Verschlucken R 36 37 38 Irritating to eyes respiratory system and skin Reizt die Augen Atmungsorgane und die Haut R 42 May cause sensitization by inhalation Sensibilisierung durch Einatmen m glich Safety phrases S 13 Keep away from food drink and animal feedstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten S 16 Keep away from sources of ignition No Smoking Von Ztindquellen fernhalten Nicht rauchen 22 Do not breathe dust Staub nicht einatmen 24 Avoid contact with the skin Ber hrung mit der Haut vermeiden S26 In case of contact with the eyes rinse with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren S 36 37 Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen 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 MACHEREY NAGEL 08 2013 Rev 01 11 DNA isolation from forensic samples 4 2 GHS classification Only harmful features do not need to be labeled with H and P phrases until 125 mL or
20. n and incubate at 56 C for at least 60 min or overnight with shaking Alternatively lysis step can be performed in Tube Strips see ordering information Following the lysis incubation spin down to collect any sample from the lysis tube lids and transfer each lysate to the wells of a Square well Block 2 Bind nucleic acid to magnetic beads Add 12 uL resuspended F Beads and 800 uL Buffer FBB to the lysed sample Mix by pipetting up and down 6 times and shake for 10 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 Be sure to resuspend the NucleoMag F Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has been formed Separate the magnetic beads against the side of the wells by placing the Square well Block on the NucleoMag SEP a magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipetting Do not disturb the attracted beads while aspirating the supernatant 3 Wash with FWB1 Remove the Square well Block from the NucleoMag SEP magnetic separator Add 600 pL Buffer FWB1 and resuspend the beads by shaking until the beads are resuspended completely 1 3 min Alternatively resuspend beads completely by repeated pipetting up and down MACHEREY NAGEL 08 2013 Rev 01 17 Nuc
21. ock and resuspend the beads by shaking 5 min at room temperature Alternatively resuspend beads completely by repeated pipetting up and down and incubate for 10 min at 72 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 DNA to either elution plates or tube strips see ordering information 18 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Insufficient elution buffer volume 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 the efficiency of following wash and elution steps Beads dried out Poor yield Do not let the beads dry as this might result in lower elution low sensitivity efficiencies Aspiration of attracted bead pellet e Do not disturb the attracted beads while aspirating the supernatant especially when the magnetic bead pellet is not visible in the lysate Aspiration and loss of beads Time for magnetic separation too short or aspiration speed too high Low purity low sensitivity Insufficient washing procedure Use only the appropriate combinati
22. ons of separator and plate for example Square well Block in combination with NucleoMag SEP Make sure that beads are resuspended completely during the washing procedure If shaking is not sufficient to resuspend the beads completely mix by repeated pipetting up and down Poor performance of RNA in downstream applications Carry over of ethanol from wash buffers e Be sure to remove all of the ethanolic Buffer FWB2 from the final wash as residual ethanol interferes with downstream applications MACHEREY NAGEL 08 2013 Rev 01 19 DNA isolation from forensic samples Poor Ethanol evaporation from wash buffers performance e Close buffer bottles tightly avoid ethanol evaporation from of DNA in buffer bottles as well as from buffer filled in reservoirs Do not downstream reuse buffers from buffer reservoirs applications continued 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 from Carry over the well of beads Aspiration speed too high elution step High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step 6 2 Ordering information Product REF Pack of NucleoMag Forensic 744650 1 1 x 96 preps 744650 4 4 x 96 preps NucleoMag SEP 744900 1 Square well Blocks 740481EO 4 ethylene oxide treated Self adhering PE Foil 740676
23. stable for at least one year All buffers are delivered ready to use Before starting any NucleoMag Forensic protocol prepare the following Proteinase K Before first use of the kit add indicated volume of Proteinase Buffer PB to each vial of the lyophilized Proteinase K Dissolved Proteinase K solution should be stored at 20 C and is stable for at least 6 months NucleoMag Forensic 1 x 96 preps 4 x 96 preps REF 744650 1 744650 4 Proteinase K 3 vials 40 mg vial 6 vials 75 mg vial lyophilized Add 1 8 mL Proteinase Buffer Add 3 35 mL Proteinase to each vial Buffer to each vial Wash Buffer FWB2 50 mL 4x50 mL Concentrate Add 200 mL ethanol Add 200 mL ethanol to each bottle 10 MACHEREY NAGEL 08 2013 Rev 01 DNA isolation from forensic samples 4 Safety instructions The following components of the NucleoMag Forensic 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 FBB Sodium perchlorate 20 40 x Xn R 10 22 S 13 16 ethanol 35 55 Natriumperchlorat 20 40 Ethanol 35 55 FWB1 Sodium perchlorate 5 20 t R 10 S 16 ethanol 20 35 Natriumperchlorat 5 20 Ethanol 20 35 Proteinase K Proteinase K lyophilized x Xn R 36 37 38 S 22 24 Proteinase K
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