Manual - Omega Bio-Tek
Contents
1. 2 Add 20 uL Proteinase K Solution 20 mg mL and incubate for 2 hours at 60 C Invert the tube occasionally to disperse the sample or place on a rocking platform 3 Add 220 uL Buffer MSL to the sample and mix by vortexing 4 Centrifuge at full gt 13 000 x g for 5 minutes 5 Transfer 400 pl sample to a new 1 5 ml tube For 96 well microplate procedure transfer 250pl of sample to each well of the microplate 6 Add 270u l for singe tube or 170pl absolute ethanol to each sample 7 Add 10pl of Mag Bind particles and mix throughly by vortexing or pipetting 10 11 12 13 14 15 16 17 18 19 20 21 22 up and down for 20 times Incubate at room temperature for 5 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles The solution should be cleared after all the magnetic beads are pelleted Carefully remove and discard the cleared supernatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 500 uL for 1 5 ml tube or 300 uL for 96 well plate SPM Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully remove and discard the cleared supe2. Life Science Typical yields from these swabs are 0 5 3 ug DNA Scrape the swabs firmly against the inside of each cheek 6 7 times Air or vacuum dry the swabs for 2 hours after collection The person providing the sample should not eat or drink for at least 30 minutes prior to the sample collection Carefully break or cut off the end part of the swab or brush into a 1 2 ml plate and add 400 uL TL to the tube Add 20 uL Proteinase K Solution 20mg ml Seal the Plate with Sealing Film Incubate 60 minutes at 56 C Transfer 280 ul of lysate into a 1 2 mL Round Well Plate compatible with MSD 0O1B Add 280 uL Buffer MSL to the sample Seal The plate with Sealing Film not provided Mix immediately by vortexing for 30 seconds Remove the Sealing film Add 380 ul Isopropanol and 10 ul Mag Bind Particles C and 10 uL of binding enhancer Incubate at room temperature for 5 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles The solution should be cleared after all the magnetic beads are pelleted Carefully remove and discard the cleared supernatant by pipetting 10 11 12 13 14 15 16 17 18 19 20 21 22 Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 300 uL MP Buffer and mix throughly by vortexing or pipetting up and down for 20 times
3. Prepare FRESH Buffer MP Ethanol as follows This mixture can only be stored at room temperature for two weeks M6225 00 Add 3 ml absolute ethanol M6225 01 Add 30 ml absolute ethanol M6225 02 Add 60 ml absolute ethanol M1427 00 Add 30 ml absolute ethanol M1427 01 Add 120 ml absolute ethanol M1427 02 Add 600 ml absolute ethanol New in this edition Proteinase K is now supplied in a liquid form eliminating the step to resuspend prior to use Proteinase K Solution can also be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in this kit Forensic DNA Protocol Protocol For Isolation of DNA From Dried Blood Body Fluids and Sperm Spots Dried blood body fluids and sperm samples on filter paper can be processed using the following method This kit can also be used for samples collected by using other specimen collection papers User Supplied Materials Centrifuge capable of 13 000 x g or 3 000 x g for 96 well plates lsopropanol 96 well magnetic stand or 1 5 mL magnetic stand 1 5 ml tube or 500 uL microplate Absolute Ethanol 96 100 An incubator capable of 70 C Deep Well of 1 2 mL round well plate Sealing film or caps for Deep well or 1 2 mL round well plate Before Starting Set an Incubator 55 C after step 2 set to 60 C Set an Incubator to 70 C Prepare Reagents according to preparing reagents section 1 Cut or punch out the blood spot or other sample from the
4. For Isolation of DNA From Saliva 10 11 12 Collect 200 uL saliva in a 1 5 mL centrifuge tube contains 200 uL Buffer MSL and 20 ul of Proteinase K Solution Mix the sample throughly by vortexing or pipetting up and down for 20 times Incubate at 65 C for 30 minutes Centrifuge at 14 000 x g for 2 minutes and transfer the sample to a new 1 5 ml tube Optional If RNA free DNA is desired add 10 ul of RNase A 25mg ml and incubate at room temperature for 5 minutes Add 10ul Mag Bind Particles followed by 290 ul of absolute ethanol Mix throughly by vortexing or pipetting up and down for 20 times Incubate at room temperature for 5 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles The solution should be cleared after all the magnetic beads are pelleted Carefully remove and discard the cleared supernatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 300 uL MP Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times Follow the standard protocol for dried blood or body fluids Page 5 from Step 13 26 Protocol For Isolation of DNA From Hair Nails and Feathers a Cut the sample into small pieces 0 5 1 cm and transfer it to a 1 5 mL centrifuge tube Tip For hair cut from base of hair for feathers select t
5. K Solution of proteinase k SPM Wash Buffer Concentrate not diluted with absolute ethanol No DNA eluted Prepare SPM Wash Buffer Concentrate as instructed on the label Problem with Insufficient DNA was 1 Use more stating material downstream used application 2 Quantify the purified DNA accurately and use sufficient DNA Excess DNA was used Make sure to use correct amount DNA for downstream application 12
6. Mag Bind particles from the magnetic separation device Add 50 200ul of Elution Buffer to the tube or each well of the microplate Mix throughly by vortexing or pipetting up and down for 50 times Incubate at 60 C for 15 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully transfer the cleared supernatant contains eluted DNA to a clean 1 5 ml tube or microplate Protocol For Isolation of Genomic DNA From Sperm This protocol can be used for fresh or frozen semen samples with equal efficiency Frozen samples must to be thawed thoroughly before use Note that lysis time will vary depending on the size and density of the source material User Supplied Materials Centrifuge capable of 13 000 x g lsopropanol 96 well magnetic stand or 1 5 mL magnetic stand 1 5 ml tube or 500 uL microplate Absolute Ethanol 96 100 An incubator capable of 70 C Make the following buffer before starting 200 mM NaCI Buffer SL 20mM Tris HCl pH 8 0 20mM EDTA pH 8 0 4 SDS 1 B mercaptoethanol Before Starting Set an Incubator 55 C after step 2 set to 60 C Set an Incubator to 70 C Prepare Reagents according to preparing reagents section 1 Add 100uL of sperm to 100uL of Buffer A in a glass Corex centrifuge tube Vortex for 10 sec at full speed Only use Corex tubes to prevent attachment of the sperm cells to the tube walls
7. Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully remove and discard the cleared supernatant by pipetting Add 400 uL of SPM Buffer and mix throughly by vortexing or pipetting up and down 20 times Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully remove and discard the cleared supernatant by pipetting Repeat Steps 13 15 Leave the tube to air dry on the magnetic separation device for 5 10 minutes Remove any residue liquid from tube by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 50 100 uL of Elution Buffer to the tube or each well of the microplate Mix throughly by vortexing or pipetting up and down for 50 times Incubate at 60 C for 15 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully transfer the cleared supernatant contains eluted DNA to aclean 1 5 ml tube or microplate Protocol for Isolation of Bacterial DNA From Biological Fluids 1 2 10 Pellet bacteria by centrifuging 10 minutes at 8 000rpm Resuspend bacterial pellet with 200 uL TL buffer Follow the protocol for dried blood body fluids and sperm spot Page 4 from Step 3 Protocol
8. filter paper Up to 200 uL of blood can be used for each spot Tear or cut filter into small pieces and place into a microfuge tube or 96 well plate A deep well or 1 2 mL round well plate can be used Note Use 3 4 punched cycles 3mm diameter for each DNA isolation 2 Add 200ul Buffer TL and 25 ul Proteinase K Solution and mix by vortexing Seal the plate with sealing film or caps Incubate for 30 45 minutes at 55 C with occasional mixing 3 Add 200ul Buffer BL and incubate at 70 C for 10 minutes Vortex every 2 min to mix Seal the plate with sealing film or caps 4 Centrifuge at maximum speed 13 000 20 000 x g for 5 minutes or 3 000 x g for 10 minutes fo r96 well plates NOTE If maximum DNA recovery is required the well of 96 well lysate clearance plate can be used to collect maximum volume of the liquid 5 Transfer 400 ul for single tube or 200ul for 96 well plate of lysate from previous step to a 1 5 ml tube or 96 well microplate 500ul 6 Add 280 ul or 140 ul of isopropanol followed by 10ul of Mag Bind particles and mix throughly by vortexing or pipetting up and down for 20 times 7 Incubate at room temperature for 5 minutes 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles The solution should be cleared after all
9. the magnetic beads are pelleted Carefully remove and discard the cleared supernatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 400 uL or 300 uL MP Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times Note It is critical to wash the magnetic particles by breaking up the magnetic particle pellet for DNA purity Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully remove and discard the cleared supernatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 500 uL or 300 uL SPM Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times Note SPM Buffer must be diluted with ethanol before use Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully remove and discard the cleared supernatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Wash the Mag Bind particles again by repeating step 16 18 with SPM Buffer Leave the tube to air dry on the magnetic separation device for 5 10 minutes Remove any residue liquid from tube by pipetting Remove the tube or plate containing the
10. Contents INtrOdUCHON iwc se fa har hee Aes EE Re ee bed wha ees OS Rd 2 PHIMCIDIS cic ta Se oN RS a pe eG Brett ah ay ee eee BY oe hol E 2 Storage and Stability secs ninpi moria ee 3 KitGontents 2 ssieiwee Awe ia the ae Baer eid Deva E Ani ek 3 Preparing Reagents 0 00000 eee 4 Protocol For Dried Body Body Fluid and Sperm Spots 5 Protocol For DNA isolation from Sperm 00005 geal Protocol For Buccal Swabs 0 00 eee ee 9 Protocol For Bacterial DNA From Biological Fluids 10 Protocol For Saliva o csc ese be ee ee Re ee ee ee ee 10 Protocol For Hair Nails and Feathers 000 11 Troubleshooting Guide 0 0000 ees 12 Revised May 2012 Introduction The E Z N A Mag Bind Forensic DNA Isolation Kit is designed to provide a rapid and easy method for the isolation of genomic DNA from forensic samples such as dry blood buccal swabs and sperm for consistent PCR and Southern analysis This kit can also be used for the preparation of genomic DNA from mouse tail snips whole blood buffy coat serum and plasma The kit allows single or multiple simultaneous processing of samples High quality genomic DNA isolated with Mag Bind technology is suitable for direct use in most downstream applications such as amplifications and enzymatic reactions This system can be easily adapted with automated system and the procedure can be scaled up or down allowing pu
11. ct M6225 00 Purification Mag Bind Particles C 55 uL 530 uL 2 2 mL patens Buffer MSL 1 Eee mL 15 mL a 60 mL Buffer TL 1 temic mL mL st j i mL SPM Buffer 2 mL 42mL___ mL 5some O mL Binding Enhancer 55 uL 550 uL 2 2 mL peee MP Buffer C mL 20 mL eme ae 40 mL Elution Buffer Buffer 2m mL 30m_ 2x50mL mL 2x 50 mL Proteinase K Solution 150 uL 1 5 mL 6 mL User Manual i a l a Product M1427 00 Purification Mag Bind Particles C i Buffer MSL Buffer TL SPM Buffer MP Buffer 400 mL Elution Buffer Binding Enhancer 1 1 mL 4 4 mL 22 mL Proteinase K Solution 3 mL 12 mL 60 mL User Manual 1 1 CAUTION Buffer MSL contains a chaotropic salt Please wear gloves and appropriate eye ware while performing this procedure NOTE The E Z N A Mag Bind Forensic DNA Isolation Kit is supplied with enough buffer for the standard protocol However due to increased volumes called for in some protocols fewer preparations may be performed Also additional buffers can be purchased separately from Omega Bio Tek See the Accessories section in the catalog or call customer service for price information Preparing Reagents SPM Buffer must be diluted with absolute ethanol as follows Important M6225 00 Add 8 mL ethanol M6225 01 Add 48 mL ethanol bottle M6225 02 Add 200 mi ethanol bottle M1427 00 Add 100 mL ethanol bottle M1427 01 Add 400 ml ethanol bottle M1427 02 Add 800 mL ethanol bottle
12. he primary feathers Large birds secondary tail or breast feather can be use Add 200 uL TL Buffer 25 uL Proteinase K Solution and 20 uL 1M DTT Mix throughly by vortexing Incubate 30 min at 60 C with occasional mixing Add 225 uL Buffer MSL to the sample mix throughly by vortexing 4 Centrifuge at maximum speed gt 14 000 x g for 5 minutes 5 Follow the protocol for sperm Page 6 from Step 5 Centrifugal Protocol Note Please read through previous sections of this manual before using this protocol 1 Prepare samples by following the standard protocol in previous sections 2 For all binding washing and elution steps Instead to use the magnetic separation device to collect the Mag Bind particles centrifuge the tube or plate at 14 000 x g for 1 minute for tube or 3000 x g for 3 minutes to collect the magnetic beads Troubleshooting Guide Problem Likely Cause Suggestions Resuspend the magnetic particles by Incomplete resuspension of magnetic particle vortexing before use Inefficient cell lysis due to inefficient mix of buffer MSL and sample mixed with BufferMSL SPM Buffer were not Prepare the SPM Buffer by adding prepared correctly ethanol according to instruction Lose of magnetic careful not remove the magnetic beads beads during opetation during the operation Make sure the sample is throughly to decrease of activity Inefficient cell lysis due Add more Proteinase
13. rification from various amounts of starting materials Principle E Z N A Mag Bind Forensic DNA Isolation Kits use the reversible binding properties of the Mag Bind paramagnetic particles to provide a fast and flexible method for isolating genomic DNA from different forensic sources Samples are first lysed with a specially formulated buffer containing detergent in the presence of Proteinase K After adjust the binding condition the sample was mixed with Mag Bind particles and the genomic DNA was bound to the surface of Mag Bind magnetic particles Proteins polysaccharides and cellular debris are efficiently washed away with few wash steps Pure DNA is then eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification Storage and Stability All components of the E Z N A Mag Bind Forensic DNA Isolation Kit except the Mag bind Particles and Binding Enhancer can be stored at 22 C 25 C Proteinase K can be stored at room temperature For long term storage gt 12 months store Proteinase K at 2 8 C Mag Bind Particles and Binding Enhancer should be stored at 2 8 C Under these conditions performance of all components of the kit are guaranteed at least 12 months Under cool ambient conditions a precipitate may form in the Buffer TL and MSL In case of such an event heat the bottle at 50 C to dissolve the precipitate Kit Contents Produ
14. rnatant by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Wash the Mag Bind particles again with SPM Buffer by repeating step 11 13 Leave the tube to air dry on the magnetic separation device for 5 10 minutes Remove any residue liquid from tube by pipetting Remove the tube or plate containing the Mag Bind particles from the magnetic separation device Add 50 200 ul of Elution Buffer to the tube or each well of the microplate Mix throughly by vortexing or pipetting up and down for 50 times Incubate at 60 C for 15 minutes Place the tube or plate to a magnetic separation device suitable for 1 5 ml tube or 96 well microplate to magnetize the Mag Bind particles Carefully transfer the cleared supernatant contains eluted DNA to aclean 1 5 ml tube or microplate Protocol For Isolation of Genomic DNA From Buccal Swabs User Supplied Materials lsopropanol 96 well magnetic stand or 1 5 mL magnetic stand 1 5 ml tube or 500 uL microplate Absolute Ethanol 96 100 An incubator capable of 70 C Deep Well of 1 2 mL round well plate compatible with MSD 01B Magnetic Stand MSD 01B Sealing film or caps for Deep well or 1 2 mL round well plate Before Starting Set an Incubator 55 C after step 2 set to 60 C Set an Incubator to 70 C Prepare Reagents according to preparing reagents section This protocol has been tested for the following swab types cotton C E P
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