MagMAX™ DNA Multi-Sample Ultra Kit (blood cards) User Guide
Contents
1. Add 200 uL of PK Mix to each sample well of a MagMAX Express 96 Deep Well Plate PK Plate Cut out 1 or 2 pieces of the blood cards 2 3 mm each using a scalpel or a hole puncher and transfer them to the appropriate wells of the PK Plate using forceps IMPORTANT Ensure that the card pieces are entirely submersed in liquid before starting PK digestion Seal the plate with a MicroAmp Clear Adhesive Film and shake the sealed plate for 10 minutes at speed 8 Incubate overnight at 65 C then shake the sealed plate for 5 minutes at speed 8 IMPORTANT Arrange plates in the incubator to allow adequate flow around the plate wells to ensure that samples quickly reach and maintain the incubation temperature 2 Set up the processing plates Just before the 65 C incubation is complete set up the Wash Elution and Tip Comb Plates outside the instrument as described in the following table MagMAX DNA Multi Sample Ultra Kit blood cards User Guide Set up the processing plates continued Add Multi Sample DNA Lysis Buffer Bead RNase Mix and Table 1 Processing plates Plate ID Plate position Plate type Reagent Volume per well Wash Plate 1 2 Deep Well Wash Solution 1 150 uL Wash Plate 2 3 Deep Well High Salt Wash Solution 150 uL Wash Plate 3 4 Deep Well Wash Solution 2 150 uL Elution Platel 5 Standard DNA Elution Buffer 1 50 pL Place a MagMAX Express 96 De2. as Cat no A25561 and DNA Binding Beads are also available as Cat no A25562 21 Final volume see Before first use of the kit prepare Wash Solutions on page 2 Materials required but not supplied Unless otherwise specified all materials are available from Life Technologies MLS major laboratory supplier For Research Use Only Not for use in diagnostic procedures Whatman FTA Elute Cards or equivalent MLS RNase free Microfuge Tubes 2 0 mL Cat no AM12425 Conical tubes 15 mL Cat no AM12500 Conical tubes 50 mL Cat no AM12502 Aerosol resistant pipette tips MLS Reagent reservoirs MLS Reagents Ethanol 200 proof absolute MLS Isopropanol 100 molecular grade or higher MLS NaCl 5M Cat no AM9760G Cat no AM9260G EDTA 0 5M pH 8 0 1 See If needed download the KingFisher Flex program on page 2 21 Available from Fisher Scientific technologies A Thermo Fisher Scientific Brand Sample collection and storage 1 Collect 40 uL of blood samples onto Whatman FTA Elute Cards using one of the following methods Note A different collection volume might be needed for other types of blood cards e Finger stick Collect samples directly on the blood cards e Venipuncture Collect samples in EDTA or sodium citrate anticoagulant tubes then transfer to blood cards Note Heparin is not recommended as an anti coagulant since it can cause inhibition of PCR rea
3. 0630 Equipment Thermo Scientific Titer Plate Shaker Cat no 14 271 9 21 One of the following incubators or an equivalent in shelves and thermometer cubator with slatted Economy Lab Incubator Cat no S50441A 21 VWR Digital Mini Incubator VWR Cat no 10055 006 Fisher Scientific Analog Vortex Mixer Cat no 02 215 365 21 Adjustable micropipettors MLS Multi channel micropipettors MLS Optional but recommended Magnetic Stand 96 Cat no AM10027 Scalpel or hole puncher MLS Forceps MLS Plastics and consumables MagMAx Express 96 Deep Well Plates Cat no 4388476 MagMAx Express 96 Standard Plates Cat no 4388475 MagMAx Express 96 Deep Well Tip Combs Cat no 4388487 MicroAmp Clear Adhesive Film Cat no 4306311 Cat no Cat no Component A25597 A25598 Storage 500 rxns 2500 rxns Proteinase K 4mL 5x4mL 15 C to 25 C PK Buffer 96 mL 5x96 mL a 15 C to 30 C Multi Sample DNA 100 mL 5 x 100 mL Lysis Buffer RNase A 2x 1 25 mL 10x 1 25mL 15 C to 25 C DNA Binding 5 Beadsll 8 mL 5x 8mL 2 C to 8 C Nuclease free Water 100 mL 5 x 100 mL Wash Solution 1 80 mL 5 x 80 mL Concentrate Wash Solution 2 162 mill 5 x 162 mL 15 C to 30 C Concentrate DNA Elution Buffer 1 25 mL 5 x 25 mL DNA Elution Buffer 2 25 mL 5 x 25 mL 1 Proteinase K is also available
4. A isolation next step 4 Wash the beads and a Select the program on the instrument elute the DNA e 4413021 DW blood on MagMAX Express 96 Magnetic Particle Processor e A25597_Blood_Buccal on KingFisher Flex Magnetic Particle Processor b Start the run and load the prepared processing plates to their positions when prompted by the instrument see Table 1 c Load the PK plate containing lysate isopropanol and Bead RNase Mix at position 1 when prompted by the instrument d When prompted by the instrument approximately 28 30 minutes after initial start 1 Remove the Elution Plate from the instrument 2 Add 50 uL of DNA Elution Buffer 2 to each sample well IMPORTANT Add DNA Elution Buffer 2 immediately after the prompt to prevent excessive drying of any beads that are still captured on the Tip Comb 3 Load the Elution Plate back onto the instrument and press Start e At the end of the run approximately 30 35 minutes after initial start remove the Elution Plate from the MagMAX DNA Multi Sample Ultra Kit blood cards User Guide instrument and seal immediately with a new MicroAmp Clear Adhesive Film e If precipitated DNA is visible pipet up and down 5 10 times before sealing the plate to ensure complete resuspension e Optional Eluates can be transferred to a storage plate after collection Wash the beads and e If excess bead residue is seen in the wells place the Elution Plate on the Magnetic Stand 96 to captur
5. USER GUIDE MagMAX DNA Multi Sample Ultra Kit app lied biosystems lik technologies High throughput isolation of PCR ready DNA from blood cards Catalog Number A25597 and A25598 Pub No MAN0010816 Rev A 0 WARNING Read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Safety Data Sheets SDSs are available from www lifetechnologies com support Product information The MagMAX DNA Multi Sample Ultra Kit is designed for rapid high throughput isolation of high quality genomic DNA from a variety of sample matrices The kit uses MagMAX magnetic bead technology ensuring reproducible recovery of PCR ready DNA suitable for a broad range of applications such as SNP genotyping and copy number experiments This protocol describes isolation of DNA from mammalian whole blood spotted onto blood storage cards optimized for use with the MagMAX Express 96 Deep Well Magnetic Particle Processor or with the KingFisher Flex Magnetic Particle Processor 96 well deep well setting The typical DNA yield obtained from 3 mm sections of card spotted with 40 uL of blood is 50 200 ng at a 0 5 2 ng L concentration Kit contents and storage Item Source Magnetic particle processor MagMAx Express 96 Magnetic Particle Processor Cat no 4400077 KingFisher Flex Magnetic Particle Processor Thermo Scientific Cat no 540
6. ctions Dry the cards at least 4 hours or according to the manufacturer s instructions lay flat and protect from moisture Process samples shortly after they are completely dry or store the cards in a dry place at room temperature Important procedural guidelines Perform all steps at room temperature 20 25 C unless otherwise noted When mixing samples by pipetting up and down avoid creating bubbles Use sterile scalpels or hole punchers and sterile forceps when preparing samples Dip them in 70 ethanol between each sample to prevent cross contamination Cover the plate during the incubation and shaking steps to prevent spill over and cross contamination The same MicroAmp Clear Adhesive Film can be used throughout the procedure unless it becomes contaminated If you use a plate shaker other than the Thermo Scientific Titer Plate Shaker verify that The plate fits securely on your titer plate shaker The recommended speeds are compatible with your titer plate shaker Ideal speeds should allow for thorough mixing without splashing Perform DNA extraction and elution Per plate volumes for reagent mixes are sufficient for one plate plus overage To calculate volumes for other sample numbers refer to the per well volume and add 5 overage If needed download the KingFisher Flex program The program required for this protocol is not pre installed on the KingFisher Flex Magnetic Particle Proce
7. e elute the DNA any residue prior to downstream use of the DNA continued IMPORTANT Do not allow the purified samples to sit uncovered at room temperature for more than 10 minutes to prevent evaporation and contamination The purified samples are ready for immediate use Alternatively store the covered Elution Plate e At2 6 C for up to 24 hours e At 20 C or 80 C for long term storage Recommended quantitation methods Note Mix the samples by pipetting up and down before quantitation ne E if they ha n stored frozen Standard curve analysis is the most accurate quantitation method if they have peen stored Dee Use the TaqMan RNase P Copy Number Reference Assay Cat Revision history no 4403326 for human genomic DNA and the TaqMan DNA Template Reagents Cat no 401970 to create a standard curve Refer Revision Date Description to Creating Standard Curves with Genomic DNA or Plasmid DNA AO September 2014 New document Templates for Use in Quantitative PCR Pub no 4371090 Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support The information in this guide is subject to change without not
8. ep Well Tip Tip Comb 6 Deep Well Comb in a MagMAX Express 96 Deep Well Plate 1 Position on the instrument 21 The instrument prompts the user to add DNA Elution Buffer 2 to the Elution Plate after incubation with DNA Elution Buffer 1 Optional If condensation is present at the end of the agitation centrifuge the plate for 1 2 minutes at 1500 x g Transfer samples to the wells of a new MagMAX Express 96 Deep Well Plate and discard the previous plate isopropanol c Add 200 uL of Multi Sample DNA Lysis Buffer to each sample d Seal the plate with the MicroAmp Clear Adhesive Film and shake for 5 minutes at speed 8 Prepare Bead RNase Mix according to the following table IMPORTANT Prepare the Bead RNase Mix up to 20 minutes before use Prolonged storage at room temperature can reduce its efficiency Vortex the DNA Binding Beads at moderate speed to form a uniform suspension before pipetting Component Volume per well Volume per plate DNA Binding Beads 16 uL 1 6 mL RNase A 5 ul 500 uL Nuclease free Water 19 uL 1 9 mL Total Bead RNase Mix 40 uL 4mL f Vortex the Bead RNase Mix at moderate speed to ensure thorough mixing and add 40 uL to each sample then use a multi channel micropipettor to mix by pipetting up and down 5 times g Seal the plate with the MicroAmp Clear Adhesive Film and shake for 5 minutes at speed 8 h Add 240 uL of isopropanol to each sample and proceed immediately to DN
9. ice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified VWR is a trademark of VWR International LLC TaqMan is a trademark of Roche Molecular Systems Inc used under permission and license Whatman and FTA are registered trademarks of GE Healthcare Inc For support visit lifetechnologies com support or email techsupportfalifetech com lifetechnologies com 15 September 2014 technologies
10. ssor 1 On the MagMAX DNA Multi Sample Ultra Kit web page scroll down to the Product Literature section 2 Right click A25597_Blood_Buccal and select Save as Target to download to your computer 3 Refer to Thermo Scientific KingFisher Flex User Manual Cat no N07669 and BindIt Software User Manual Cat no N07974 for instructions for installing the program on the instrument Before first use of the kit prepare Wash Solutions 1 Prepare the Wash Solutions from the concentrates e Add 25 mL of isopropanol to Wash Solution 1 Concentrate mix and store at room temperature e Add 132 mL of ethanol to Wash Solution 2 Concentrate mix and store at room temperature Prepare High Salt Wash Solution as indicated in the following table mix and store at room temperature Component Volume dane 5M NaCl 40 mL 2M 0 5 M EDTA pH 8 0 4 uL 2 mM Ethanol 40 mL 40 v v Water up to 100 mL Total High Salt Wash Solution 100 mL 1 Digest the samples with a Proteinase K mix components Preheat an incubator to 65 C b Prepare sufficient PK Mix according to the following table Invert PK Mix several times to thoroughly IMPORTANT Prepare the PK Mix just before use Do not place PK Buffer or PK Mix on ice to avoid precipitation Component Volume per well Volume per plate Proteinase K 8 uL 800 uL PK Buffer 192 uL 19 2 mL Total PK Mix 200 uL 20 mL
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