Genomic DNA from Blood
Contents
1. Genomic DNA from Blood User Manual NucleoMag Blood 3 mL September 2010 Rev 01 MACHEREY NAGEL MN Genomic DNA from Blood Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Material to be supplied by the user 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Elution procedures 6 3 Storage conditions and preparation of working solutions 7 4 Safety instructions risk and safety phrases 8 5 Standard procedure for the isolation of genomic DNA from 3 mL blood samples using KingFisher Flex 24 9 6 Appendix 11 6 1 Troubleshooting 11 6 2 Ordering information 12 6 3 Product use restriction warranty 13 MACHEREY NAGEL 09 2010 Rev 01 3 Genomic DNA from Blood 1 Components 1 1 Kit contents NucleoMag Blood 3 mL 1x 96 preps REF 744502 1 NucleoMag B Beads 18 mL Lysis Buffer MBL1 125 mL Binding Buffer MBL2 550 mL Wash Buffer MBL3 1000 mL Wash Buffer MBL4 500 mL Elution Buffer MBL5 125 mL Proteinase K lyophilized 12x 75 mg Proteinase Buffer PB 2x 35 mL User Manual 1 1 2 Material to be supplied by the user e Magnetic separator KingFisher Flex 24 instrument e Separation plates elution plates KingFisher 24 deep well plates e KingFisher 24 well tip comb e 80 ethanol for the washing step Elution Buffer MBL5 5 mM Tris pH 8 5 For preparation of working solutions and storage conditions see section 3 4 MACHEREY2. 42 May cause sensitization by inhalation Safety phrases S 22 Do not breathe dust S 24 Avoid contact with the skin S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S 36 37 Wear suitable protective clothing and gloves Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not necessary if quantity per bottle below 25g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 8 MACHEREY NAGEL 09 2010 Rev 01 NucleoMag Blood 3 mL 5 Standard procedure for the isolation of genomic DNA from 3 mL blood samples using KingFisher Flex 24 The script necessary to run the NucleoMag Blood 3 mL kit on the KingFisher Flex 24 is available through MN technical support Lyse sample Prepare KingFisher 24 deep well plate with buffers label deep well blocks before use Wash and elution buffers Fill 1 mL Elution Buffer MBL5 to each well of an empty Thermo KingFisher 24 deep well plate Fill 4 8 mL Wash Buffer MBL4 to each well of an empty Thermo KingFisher 24 deep well plate Fill 4 8 mL 80 ethanol to each we
3. in Buffer MBL4 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 Incubation after dispensing beads to lysate e Mix immediately after dispensing NucleoMag B Beads and Binding Buffer MBL2 to the lysate Poor blood quality e Be sure that no blood clots are transferred to the lysis plates Blood can be stored at 2 8 C for two weeks Freeze samples if stored for longer periods Low purity Incomplete magnetic bead separation e High amounts of eluted DNA increase the viscosity and prevent the beads from being attracted completely to the magnets Increase elution buffer volume MACHEREY NAGEL 09 2010 Rev 01 11 Genomic DNA from Blood Problem Possible cause and suggestions Carry over of ethanol from ethanol wash step e Be sure to remove all of the ethanol from the ethanol wash Suboptimal step Carry over of ethanol may interfere with downstream performance applications Typically washing the beads in Buffer MBL4 is of DNA in sufficient to remove ethanol However if necessary include a downstream 10 min airdrying step following the Buffer MBL4 wash step 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 magnets Carry over of beads Incomplete magnetic bead separation e High amounts of eluted DNA increase the viscosity a
4. NAGEL 09 2010 Rev 01 Genomic DNA from Blood 2 Product description 2 1 The basic principle The NucleoMag Blood 3 mL 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 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 for downstream applications NucleoMag Blood 3 mL is recommended for use on KingFisher Flex 24 instrument 2 2 Kit specifications The NucleoMag Blood 3 mL kit is made for isolation of genomic DNA from blood samples This kit provides reagents and magnetic beads for isolation of genomic DNA from 96 samples of up to 3 mL The purified DNA can be used directly as template for PCR blotting or any kind of enzymatic reactions The kit provides reagents for the purification of up to 100 130 ug of pure genomic DNA from 3 mL whole blood with anA A ratio 1 6 1 9 260 280 Fresh frozen or blood treated either with EDTA or citr
5. ate can be used NucleoMag Blood 3 mL kit can be processed completely at room temperature Elution at 55 C will increase the yield by about 15 20 NucleoMag B Beads are highly reactive superparamagnetic beads with a high binding capacity NucleoMag Blood 3 mL kit has been developed for use with ThermoFisher s KingFisher Flex 24 instrument A script is available on request from MACHEREY NAGEL The maximum sample volume of 3 mL is splitted into two aliquots of 1 5 mL each For processing smaller blood sample volumes use of liquid handling robots other than the KingFisher Flex 24 or manual extraction please inquire with MN technical support for details For smaller blood sample volumes MN offers the NucleoMag Blood 200 uL kit see ordering information MACHEREY NAGEL 09 2010 Rev 01 5 Genomic DNA from Blood 2 3 Elution procedures Purified genomic DNA can be eluted directly with the supplied Elution Buffer MBL5 Elution can be carried out in a volume of gt 1 mL Smaller elution buffer volumes may result in incomplete bead separation For efficient elution the magnetic bead pellet should be resuspended completely in the elution buffer MACHEREY NAGEL 09 2010 Rev 01 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 e All components of the NucleoMag Blood 3 mL
6. clusive 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 MACHEREY NAGEL 09 2010 Rev 01 13 Genomic DNA from Blood 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 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 Trademarks KingFisher is a registered trademark of Thermo Fish
7. e 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 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 ex
8. er Scientific NucleoMag is a registered trademark of MACHEREY NAGEL GmbH amp Co KG 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 14 MACHEREY NAGEL 09 2010 Rev 01
9. kit should be stored at room temperature 18 25 C and are stable for up to one year e All buffers are delivered ready to use Before starting NucleoMag Blood 3 mL protocol prepare the following e 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 3 mL 1 x 96 preps REF 744502 1 Proteinase K Add 3 75 mL Proteinase Buffer PB to each vial MACHEREY NAGEL 09 2010 Rev 01 7 Genomic DNA from Blood 4 Safety instructions risk and safety phrases The following components of the NucleoMag Blood 3 mL 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 MBL1 Guanidinium x Xn Harmful if swallowed R 22 36 38 S 22 hydrochloride Irritating to eyes and skin MBL2 Sodium Flammable R10 perchlorate ethanol lt 50 MBL3 Sodium per Flammable R 10 chlorate lt 15 ethanol lt 24 Proteinase K Proteinase K x Xn Irritating to eyes R 36 37 38 S 22 24 lyophilized Xi respiratory system 42 26 36 37 and skin May cause sensitization by inhalation Risk phrases R 10 Flammable R 22 Harmful if swallowed R 36 37 38 Irritating to eyes respiratory system and skin R 36 38 Irritating to eyes and skin R
10. ll of an empty Thermo KingFisher 24 deep well plate Fill 4 8 mL Wash Buffer MBL3 to each well of an empty Thermo KingFisher 24 deep well plate Fill 4 8 mL Wash Buffer MBL3 to each well of a second empty Thermo KingFisher 24 deep well plate Fill 150 pL of Proteinase K working solution to each well of the two lysis plates Thermo KingFisher 24 deep well plates Please note that 3 mL blood samples have to be split and distributed into two plates 1 5 mL for each plate Fill 1 5 mL blood sample to a well of the lysis plate Thermo KingFisher 24 deep well plate with 150 uL Proteinase K per well Fill 1 5 mL blood of the same sample to the well at the same position of the second lysis plate Make sure that one sample is distributed into the same position of each deep well plate e g sample 1 to position A1 of lysis plate 1 and position A1 of lysis plate 2 sample 2 to position A2 of lysis plate 1 and position A2 of lysis plate 2 etc MACHEREY NAGEL 09 2010 Rev 01 9 NucleoMag Blood 3 mL After adding the samples add 575 uL Buffer MBL1 to each well of the two lysis plates Start isolation on King Fisher Flex 24 instrument Start method NucleoMag _Blood_3mL method is available from MN on request Insert plates as indicated on the KingFisher instrument display Method starts with a mixing step sample lysis after setting up the last plate to the instrument After mixing steps for lysi
11. nd prevent the beads from being attracted completely to the magnets Increase elution buffer volume Cross conta mination Overtilling of wells from the 24 well separation plate e Do not overfill the wells of the separation plates to avoid cross contamination by splashing 6 2 Ordering information Product REF Pack of NucleoMag Blood 3 mL 744502 1 1 x 96 preps NucleoMag Blood 200 uL 744501 1 1 x 96 preps 744501 4 4 x 96 preps Visit www mn net com for more detailed product information 12 MACHEREY NAGEL 09 2010 Rev 01 Genomic DNA from Blood 6 3 Product use restriction warranty NucleoMag Blood 3 mL kit components were developed designed distributed 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 Blood 3 mL 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 th
12. s approx 10 min the instrument will ask for addition of Buffer MBL2 and NucleoMag B Beads Addition of Binding Buffer MBL2 and NucleoMag B Beads to lysis plate 1 Add 2 3 mL Buffer MBL2 and 150 pL NucleoMag B Beads to each well of the lysis plate 1 Mix up NucleoMag B Beads before use Return lysis plate 1 to the instrument and continue Addition of Binding Buffer MBL2 to lysis plate 2 Add 2 3 mL Buffer MBL2 to each well of the lysis plate 2 Return lysis plate 2 to the instrument and continue All further steps are now processed without further user interaction Remove eluted DNA The instrument stops after the final elution step Follow the instructions on instrument display and unload the plates from the instrument Purified DNA should be centrifuged before UV measurement MACHEREY NAGEL 09 2010 Rev 01 Genomic DNA from Blood 6 Appendix 6 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 subsequent washing and elution steps Beads dried out Poor DNA e Do not let the beads dry as this might result in lower elution yield efficiency Partial elution in Wash Buffer MBL4 already e Do not resuspend beads
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