NucleoMag® 96 Blood
Contents
1. Separation is carried out in a 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 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 OberschleiRheim 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 6 MACHEREY NAGEL 02 2003 Rev 01 Genomic DNA from Blood 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 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 through2. 100 ul Blood 2 Lyse cells 125 ul MB1 shake 5 min RT 3 Bind DNA to NucleoMag Blood Beads 14 ul B Beads 360 yl MB2 shake 5 min RT 4 Remove supernatant 2 min separation 5 MB3 wash step 600 ul MB3 shake 5 min RT 2 min separation Coo Coo 10 MACHEREY NAGEL 02 2003 Rev 01 NucleoMag 96 Blood 6 MB4 wash step 6 600 ul MB4 shake 5 min RT 2 min separation I pitt en etre S Lu fut 7 MB5 wash step 6 600 ul MB5 90 sec incubation 2 min separation Sara ett a et 5 dy ri jot 8 Elute genomic DNA and transfer to Elution Plate 25 100 ul MB6 6 shake 5 min RT 2 min separation transfer Optional Elution at 55 C i _ ight gh eerie amp Sr pit MACHEREY NAGEL 02 2003 Rev 01 11 NucleoMag 96 Blood 4 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 Transfer 100 ul blood 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 Add 125 ul Ly
3. Blood 5 Add 600 ul Wash Buffer MB3 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 Aspirate and discard the supernatant Remove the Square well Block from the magnetic separator Note Supernatant has a brownish color magnetic bead pellet is visible now 6 Add 600 ul Wash Buffer MB4 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 Aspirate and discard the supernatant Do not remove the Square well Block from the magnetic separator Note Supernatant is colorless magnetic bead pellet is clearly visible 7 Add 800 ul Wash Buffer MB5 to each well and incubate for 90 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 MB 5 This step is to remove traces of ethanol and eliminates a drying step 8 Add desired volume of Elution Buffer MB6 25 100 ul to each well and resuspend 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 again
4. AGEL 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 Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 and 275 e mail TECH BlIO mn net com MACHEREY NAGEL 02 2003 Rev 01 17
5. Genomic DNA from Blood User manual NucleoMag 96 Blood February 2003 Rev 01 MACHEREY NAGEL DE Genomic DNA from Blood Table of contents 1 Kit contents 2 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 Elution procedures 3 Storage conditions and preparation of working solutions 4 General procedure 4 1 Standard protocol for the purification of genomic DNA 5 Appendix 5 1 Troubleshooting 5 2 Ordering information 6 4 Product Use Restriction Warranty MACHEREY NAGEL 02 2003 Rev 01 Oo ANN DD A A A a k KR N O E C SE S ao OQO A Genomic DNA from Blood 1 Kit contents 1 x 96 preps 4 x 96 preps 24 x 96 preps 744 500 1 744 500 4 744 500 24 NucleoMag B Beads 1 68 ml 6 7 ml 40 3 ml Lysis Buffer MB1 15 ml 60 ml 360 ml Binding Buffer MB2 40 ml 2x 80 ml 8 x 120 ml Wash Buffer MB3 66 ml 264 ml 2 x 792 ml Wash Buffer MB4 66 ml 264 ml 2 x 792 ml Wash Buffer MB5 102 ml 408 ml 3 x 816 ml Elution Buffer MB6 12 ml 48 ml 288 ml Elution plate U bottom including one Self 1 4 24 adhering PE foil Protocol Material to be supplied by user Separation plate e g MN Square well Block see ordering information 4 MACHEREY NAGEL 02 2003 Rev 01 Genomic DNA from Blood 2 Product description 2 1 The basic principle The NucleoMag 96 Blood procedure is based on reversible adsorpt
6. after dispensing NucleoMag B Beads Binding Buffer MB2 to the lysate Poor blood quality e 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 Row purity Insufficient washing procedure e Use only the appropriate combinations of separator and plate e g MN Square well Block in combination with NucleoMag SEP 14 MACHEREY NAGEL 02 2003 Rev 01 Genomic DNA from Blood Problem Possible cause and suggestions Carry over of ethanol from wash buffer MB4 Suboptimal e Be sure to remove all of the ethanolic Wash Buffer MB4 performance as residual ethanol interferes with downstream of DNA in applications downstream applications Low purity e see above Carry over of beads Time for magnetic separation too short e Increase separation time to allow the beads to be completely attracted to the magnetic pins before 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 Cross contamination Contamination of the rims e 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 02 2003 Rev 01 15 Gen
7. ing 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 MACHEREY NAGEL 02 2003 Rev 01 7 Genomic DNA from Blood 8 channel pipetting device 2 6 Elution procedures Method Resuspension Speed Small elution Number of tips Efficiency volume possible needed Magnetic mix Low Shaker t Low Pipetting s High Purified genomic DNA can be eluted directly with the supplied Elution Buffer MB6 Elution can be carried out in a volume of 2 25 ul It is essential to cover the NucleoMag Blood Beads completely with elution buffe
8. ion of nucleic acids to paramagnetic beads under appropriate buffer conditions Whole blood is lysed with Lysis Buffer MB1 without a proteinase K digestion Adjusting the binding conditions of nucleic acid with Binding Buffer MB2 and addition of paramagnetic beads can be carried out simultaneously After magnetic separation and removal of 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 MBS Finally highly purified DNA is eluted with low salt Elution Buffer MB6 and can directly be used for downstream appli cations The NucleoMag 96 Blood kit can be used either manually or automated on standard liquid handling instruments g DNA A Contaminants NucleoMag Beads Whole blood is lysed with Lysis Buffer MB1 Binding conditions are adjusted and the NucleoMag Blood Beads are added to the sample DNA is bound to the NucleoMag Blood Beads Beads are held back in the well while contaminants are washed away B C 4 gt Dra DNA is eluted from the beads and recovered while beads are held back in the well by the magnet DNA S Bj is ready to use in downstream applications MACHEREY NAGEL 02 2003 Rev 01 5 Genomic DNA from Blood 2 2 Kit specifications NucleoMag 96 Blood is designed for rapid manual and automated small scale preparation of highly pure genomic DNA from whole blood
9. omic DNA from Blood 5 2 Ordering information Product Cat No Pack of NucleoMag 96 Blood 744 500 1 1 x 96 NucleoMag 96 Blood 744 500 4 4 x 96 NucleoMag 96 Blood 744 500 24 24 x 96 NucleoMag SEP 744 900 1 Square well Blocks 740 670 20 Self adhering PE foil 740 676 50 sheets 6 4 Product Use Restriction Warranty NucleoMag 96 Blood 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 Blood 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 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 an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport ins
10. r 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 5 4 Yield ug wo N 75 100 125 150 175 200 Elution Volume pl Yield does not depend on elution volume Averages of 8 samples per elution volume are shown Yield ug N wo A oO N 3 4 5 Sample Number room temperature E 55 degrees C Elution is possible at room temperature Yield can be increased by 15 20 if elution is performed at 55 C MACHEREY NAGEL 02 2003 Rev 01 Genomic DNA from Blood 3 Storage conditions and preparation of working solutions Attention Buffers MB1 MB2 MB3 and MB4 contain chaotropic salt Wear gloves and goggles Upon storage especially at low temperatures a white precipitate may form in buffer MB3 Dissolve such precipitates by incubation of the bottle at 37 C before use All components of the NucleoMag 96 Blood 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 MACHEREY NAGEL 02 2003 Rev 01 9 NucleoMag 96 Blood A General procedure 1 Transfer blood to Square well Block
11. sis Buffer MB1 to each sample and mix by shaking 5 min at room temperature Alternatively pipette up and down 10 times and incubate 5 min at room temperature For 96 samples mix at least 1344 ul of NucleoMag B Beads with 34 56 ml of buffer MB2 vortex briefly and add 374 ul of this mixture 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 B Beads and Binding Buffer MB2 should be premixed Per well to be processed mix 14 ul of NucleoMag B Beads with 360 ul Binding Buffer MB2 Vortex briefly Depending on the dead volume of the reservoir additional amounts of bead suspension and binding buffer are necessary Be sure to resuspend the NucleoMag B Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous suspension has formed 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 to 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 MACHEREY NAGEL 02 2003 Rev 01 NucleoMag 96
12. st 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 02 2003 Rev 01 13 Genomic DNA from Blood 5 Appendix 5 1 Troubleshooting Problem 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 decrease 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 MB5 already ee DNA e Keep the beads on the magnet while dispensing Wash y Buffer MB5 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
13. the buffer during the wash and elution steps Separation takes place when the system stops 2 4 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 2 5 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 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 aspirat
14. urance 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 REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS 16 MACHEREY NAGEL 02 2003 Rev 01 Genomic DNA from Blood 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 N
15. 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 96 Blood allows easy automation on common liquid handling 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 1 4 ug of pure genomic DNA from 100 ul whole blood with an A260 280 ratio 2 1 6 1 9 and typical concentration of 20 40 ng ul Depending on health status of the donor and the elution volume used concentrations of 10 160 ng ul can be obtained Fresh frozen or blood treated either with EDTA citrate or heparin can be used The procedure is optimized for a sample volume of 100 ul NucleoMag 96 Blood can be processed completely at room temperature Elution at 55 C will increase the yield by about 15 20 NucleoMag Blood Beads are highly reactive superparamagnetic beads The binding capacity is 0 4 ug of gDNA per 1 ul of NucleoMag Blood Bead Suspension 1 ul of suspension contains 130 yg of beads 2 3 Magnetic separation systems For use of NucleoMag 96 Blood the NucleoMag SEP is recommended
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