- LiStarFish
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1.2. or down allowing purification from various amounts of starting materials Principle Mag Bind Forensic DNA Isolation KF96 Kit 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 Mag Bind Forensic DNA Isolation KF96 Kit except the Proteinase K can be stored at 22 C 25 C Once reconstituted in water Proteinase K must be stored at 20 C Under these conditions performance of all components of the kit are guaranteed at least 18 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 Product M1429 00 M1429 01 M1429 02 Purification 1x96 4x96 20 x 96 Mag Bind Particles S 1 1 mL 4 2 mL 21 mL Buffer MSL 3
3. 5 mL 140 mL 700 mL Buffer TL 45 mL 175 mL 875 mL SPM Buffer 144 mL 3 x 300 mL MP Buffer 20 mL 80 mL 400 mL Elution Buffer 15 mL 60 mL 2 x 150 mL Proteinase K 60 mg 240 mg 1 2g Proteinase Storage Buffer 4 mL 15 mL 65 mL 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 Before Starting Reconstitute Proteinase K with Proteinase Storage Buffer to final concentration at 20mg ml Store at 20 C Vortex vial briefly prior to use SPM Buffer must be diluted with absolute ethanol as follows M1429 00 Add 84 mL ethanol bottle Important M1429 01 Add 336ml ethanol bottle M1429 02 Add 700 mL ethanol bottle Prepare FRESH Buffer MP Ethanol as follows This mixture can only be stored at room temperature for two weeks M1429 00 Add 30ml absolute ethanol M1429 01 Add 120ml absolute ethanol M1429 02 Add 600 mi absolute ethanol Forensic DNA Protocol KingFisher 96 Process Pipetting Instruction for KingFisher 96 and Mag Bind Forensic DNA p
4. Contents IntrOduCtION A 0 eee eye Pee eA ee A ee ea ea 2 Principle te care tetea Ca peg eos hates Bae wie en Pa 2 Storage and Stability gt Les a aoe iihi i eard aa a A eee 3 Kit Contents seme cesininui 04 e i aa i ye E a Se ee ee A 3 Before Starting n 05 545 na a he eed bei eee ee eee debe 4 Forensic DNA Protocol 0 0 0 0 eee eee 4 Protocol For Dried Body Body Fluid and Sperm Spots 4 Protocol For DNA isolation from Sperm 0 0 2 0000 eee eee 5 Protocol For Buccal Swabs 0 00 eee ee 5 Protocol For Bacterial DNA From Biological Fluids 6 Protocol For Saliva teaa a aeia ees 6 Protocol For Hair Nails and Feathers 7 Determination of Yield and Quality 0 0000 eee eee 7 Troubleshooting Guides si seeno sore yag d ari aoaaa eee 8 Revised June 2009 Introduction The Mag Bind Forensic DNA Isolation KF96 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 by using Kingfisher instrument This kit can also be used for the preparation of genomic DNA from mouse tail snips whole blood buffy coat serum and plasma 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
5. e at room temperature for 5 minutes Add 10ul Mag Bind Particles followed by 290 ul of absolute ethanol Mix throughly This will be plate 1 in Kingfisher Protocol Press start on Kingfisher 96 Saliva protocol and load plates accordingly Protocol For Isolation of DNA From Hair Nails and Feathers 1 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 the primary feathers Large birds secondary tail or breast feather can be use 2 Add 400 L TL Buffer 25 i L Proteinase K and 20 L 1M DTT Mix throughly by vortexing Incubate 30 min at 60 C with occasional mixing 3 Transfer 290 ul of lysate into a KingFisher Deep Well Plate without removing insoluable debri It may be necessary to centrifuge at 4 000 x g to pellet debri 4 Add 3001 L Buffer MSL to the sample Mix immediately by vortexing for 30 seconds Add 400 ul ethanol and 10 ul magnetic beads This will be plate 1 in the KF protocol 5 Press start on Kingfisher 96 Buccal Swabs protocol and load plates accordingly Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water Tris HCl buffer or Elution Buffer as blank DNA concentration is calculated as DNA Absorbance x 0 05 ug uL x Dilution factor The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm A ratio o
6. es to the KF Deep well plate This will be Plate 1 in Kingfisher Protocol 7 Press start on Kingfisher 96 dried blood spots protocol and load plates accordingly 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 Make the following buffer before starting 200 mM NaCl Buffer SL 20mM Tris HCl pH 8 0 20mM EDTA pH 8 0 4 SDS 1 amp mercaptoethanol 1 Add 100i L of sperm to 100i L 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 2 Add 2071 L Proteinase K 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 L Buffer MSL to the sample and mix by vortexing 4 Transfer 400 pl sample to a KF 96 Deep Well Plate 5 Add 270 pl absolute ethanol to each sample 6 Add 10ypl of Mag Bind particles and mix throughly This will be Plate 1 in Kingfisher Protocol 7 Press start on Kingfisher 96 Sperm protocol and load plates accordingly Protocol For Isolation of Genomic DNA From Buccal Swabs This protocol has been tested for the following swab types cotton C E P Life Science Typical yields from these swabs a
7. f A A Of 1 7 1 9 corresponds to 85 95 purity Expected yields vary with both amount and type of tissue used 30 mg of fresh tissue will yield 10 40 g DNA with two elutions each 200 iL Troubleshooting Guide Problem Likely Cause Suggestions i Low DNA Incomplete yields resuspension of magnetic particle Inefficient cell lysis due to inefficient mix of buffer MSL and sample SPM Buffer were not prepared correctly Lose of magnetic beads during opetation Inefficient cell lysis due to decrease of activity of proteinase k No DNA eluted SPM Wash Buffer Concentrate not diluted with absolute ethanol Resuspend the magnetic particles by vortexing before use Make sure the sample is throughly mixed with BufferMSL ethanol according to instruction careful not remove the magnetic beads during the operation Add more proteinase K solution Prepare SPM Wash Buffer Concentrate as instructed on the label Problem with Insufficient DNA was downstream used application Excess DNA was used for downstream application 1 Use more stating material 2 Quantify the purified DNA accurately and use sufficient DNA Prepare the SPM Buffer by adding Make sure to use correct amount DNA Distribuito in ITALIA da Li StarFish S r l Via Cavour 35 20063 Cernusco S N MI telefono 02 92150794 fax 02 92157285 PRODOTTI PER LA RICERCA METNCA info listarfish it www listarfish it
8. re 0 5 3 g DNA 1 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 microcentrifuge tube plate and add 400 iL TL to the tube Add 20 iL Proteinase K solution 20mg ml Incubate 60 minutes at 56 C Transfer 290 ul of lysate into a KingFisher Deep Well Plate Add 300 L Buffer MSL to the sample Mix immediately by vortexing for 30 seconds Add 400 ul ethanol and 10 ul magnetic beads This will be plate 1 in the KF protocol Press start on Kingfisher 96 Buccal Swabs protocol and load plates accordingly Protocol for Isolation of Bacterial DNA From Biological Fluids 1 2 Pellet bacteria by centrifuging 10 minutes at 8 000rpm Resuspend bacterial pellet with 200 L TL buffer Follow the protocol for dried blood body fluids and sperm spot Page 4 from Step 3 Protocol For Isolation of DNA From Salvia 1 Collect 200 L saliva in a 1 5 mL tube plate contains 200 L Buffer MSL and 20 i I of Proteinase K Mix the sample throughly by vortexing or pipetting up and down for 20 times Incubate at 65 C for 30 minutes Transfer the sample to a KF 96 Deep Well Plate Optional If RNA free DNA is desired add 10 ul of RNase A 25mg ml and incubat
9. rotocol Plate Type Plate Content Reagent Volume A 1 Sample Lysate according to protocols below A 2 MP Buffer 500 ul A 3 SPM Buffer 600 ul A 4 SPM Buffer 600 ul B 5 Elution Buffer 100 ul A KingFisher 96 DW Plate B KingFisher 96 KF Plate Prepare lysate by following the use instruction based on sample type Add 500 ul MP Buffer to Plate 2 Add 600 ul SPM Wash Buffer to Plate 3 Add 600 ul SPM Wash Buffer to Plate 4 Add 100 ul Elution Buffer to Plate 5 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 We recommend using OB Specimen Paper OSP 01 and OSP 02 for spotting blood This kit can also be used for samples collected by using other specimen collection papers such as FTA cards 1 Cut or punch out the blood spot or other sample from the filter paper tul 10ul of blood can be used for each spot Tear or cut filter into small pieces and place into a microfuge tube Note Use 3 4 punched cycles 3mm diameter for each DNA isolation 2 Add 100ul Buffer TL and 10ul Proteinase K and mix by vortexing Incubate for 30 45 minutes at 55 C with occasional mixing 3 Add 110ul Buffer MSL and incubate at 55 C for 10 minutes Vortex every 2 min to mix 5 Transfer 200 yl to a Kingfisher DW Plate 6 Add 160i of Ethanol 96 100 I followed by 101 of Mag Bind particl
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