Contents Introduction Principle Storage and Stability
Contents
1. 95 purity Expected yields vary with both amount and type of tissue used 30 mg of fresh tissue will yield 10 40 ug DNA with two elution each 200 ul Troubleshooting Guide Use the table below to find solutions to any problems you may have with the E Z N A Forensic DNA Isolation Kit Possible Cause Suggestions Clogged Column Incomplete lysis Extend incubation time of lysis with Buffer STL and protease Add the correct volume of Buffer BL and incubate for specified time at 70 C It may be necessary to extend incubation time by 10 min Sample too large If using more than 30 mg tissue increase volumes of OB Protease or Proteinase K Buffer STL Buffer BL and ethanol Pass aliquots of lysate through one column successively Sample too viscous Divide sample into multiple tubes adjust volume to 250 ul with 10 mM Tris HCl Poor sample Incubate the OB specimen release from collection paper longer in STL collection paper buffer Shake the tubes frequently Poor elution Repeat elution or increase elution volume see note on Page 4 Incubation of column at 70 C for 5 min with Elution Buffer may increase yields Improper washing Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on Page 4 before use 10 Problem Possible Cause Suggestions Low Ayeo Avgo Extended Resin from the column may be ratio centrifugation present in eluate Avoid during elut2. OSP 02 for isolation of genomic DNA from forensic samples such as dry blood and sperm This kit can be also used for fresh or frozen tissue samples or mouse tail snips call customer service for detailed protocol Principle E Z N A Forensic DNA Kit uses the reversible binding properties of the HiBind matrix a new silica based material combined with the speed of mini column spin technology A specifically formulated buffer system allows genomic DNA up to 50 kb to bind to the matrix Samples are first lysed under denaturing conditions and then applied to the HiBind spin columns to which the DNA binds while cellular debris hemoglobin and other proteins are effectively washed away High quality DNA is finally eluted in sterile deionized water or low salt buffer Each HiBind column can bind approximately 100 ug DNA Use of more than 30 mg tissue or 10 cells is not recommended Storage and Stability All components of the E Z N A Forensic DNA Kit except the OB Protease can be stored at 22 C 25 C Once reconstituted in water OB Protease must be stored at 20 C Under these conditions performance of all components of the kit are guaranteed atleast 18 months Under cool ambient conditions a precipitate may form in the Buffer BL In case of such an event heat the bottle at 37 C to dissolve the precipitate Store Buffer BL at room temperature Kit Contents Standard Protocol For Isolation of DNA From Dried Blood Body F
3. Contents Introduction Principle New In This Addition Storage and Stability Kit Contents Before Starting Protocol For Dried Body Fluid Samples 2 00000 eee eue Protocol For DNA isolation from Sperm 0 200000 e eee eee Protocol For Buccal Swab 0 0 00 cee ees Protocol For Bacterial DNA From Biological Fluids Protocol For Genomic DNA From Eye Nasal and Other Swabs Vacuum Spin Protocol Determination of Yield and Quality 0 0 0 0 eee eee Troubleshooting Guide Revised August 2005 Introduction The E Z N A Forensic DNA 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 There is no need for phenol chloroform extractions and time consuming steps such as precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A Forensic DNA method is ready for applications such as PCR Southern blotting and restriction digestion The E Z N A Forensic DNA Kit is specially designed to work with the OB Specimen Collection Paper product OSP 01 amp
4. bation at 70 C rather than at room temperature will give a modest increase in DNA yield per elution Alternatively use of the first eluate for second elution will increase DNA concentration Protocol for Isolation of Bacterial DNA From Biological Fluids 1 Pellet bacteria by centrifuging 10 minutes at 8 000rpm 2 Resuspend bacterial pellet with 200 ul STL buffer 3 Follow the standard protocol Page 4 from Step 3 Protocol For Isolation of Genomic DNA From Eye Nasal And Other Swabs 1 Collect the sample and put into 2 ml PBS Incubate 2 3 hours at 30 C 2 Pellet bacteria by centrifuging 10 minutes at 8 000rpm 3 Resuspend bacterial pellet with 200 ul STL buffer 4 Follow the standard protocol Page 4 from Step 3 Protocol For isolation of Genomic DNA from other Forensic Sample 1 Collect the sample and put into tube 2 Resuspend sample with 200 ul STL buffer 3 Add 25 ul OB protease solution and mix by votexing Incubate at 55 C in a shaking waterbath to effect complete lysis If no shaking waterbath is available vortex the sample every 20 30 minutes Lysis time depends on amount and type of samples but is usually under 3 hours One can allow lysis to proceed overnight 4 Centrifuge at 15 000 x g for 5 min and transfer the supernatant into a new tube 5 Follow the standard protocol Page 4 from Step 4 Forensic DNA Kit Vacuum Spin Protocol Note Please read through previous sections ofthis manual b
5. efore using this protocol 1 Prepare samples by following the standard protocol in previous sections Steps 1 5 Prepare the vacuum manifold according to manufacturer s instructions and connect the V Spin column to the manifold Load the sample Buffer BL ethanol mixture into the column Switch on vacuum source to draw the sample through the column then turn off the vacuum Wash the column by adding 500 ul Buffer HB Draw Buffer HB through the column by turning on the vacuum source Wash the column by adding 700 uI DNA Wash Buffer Draw the wash buffer through the column by turning on the vacuum source Repeat this step with another 700 ul DNA wash buffer Insert the column in a 2 ml collection tube Then centrifuge 1 minute to dry the column Drying the column is critical for removal of residual ethanol that might otherwise interfere with downstream applications Place the column in a nuclease free 1 5 ml microcentrifuge tube and add 30 50ul TE or water Allow to stand for 1 2 minutes then centrifuge 1 minute to elute DNA 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 of Ajgo Aogo Of 1 7 1 9 corresponds to 85
6. ion step centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests Problem Possible Cause Suggestions No ethanol added Dilute Wash Buffer with the to Wash Buffer indicated volume of absolute Concentrate ethanol before use Poor cell lysis due to incomplete mixing with Buffer BL Repeat the procedure this time making sere to vortex the sample with Buffer BL immediately and completely Increase incubation time with Buffer STL and protease Ensure that no visible pieces of tissue remain Incomplete cell lysis or protein degradation due to insufficient incubation Samples are rich in protein After applying to column wash with 300 ul of a 1 1 mixture of Buffer BL and ethanol and then with DNA Wash Buffer No DNA eluted Poor cell lysis due to improper mixing with Buffer BL Mix thoroughly with Buffer BL prior to loading HiBind column Poor cell and or protein lysis in Buffer STL Tissue sample must be cut or minced into small pieces Increase incubation time at 65 C with Buffer STL to ensure that tissue is completely lysed Absolute ethanol not added to Buffer BL Before applying sample to column an aliquot of Buffer BL ethanol must be added See protocol above No ethanol added to Wash Buffer Conce
7. luids and Sperm Spots Eroduct Number 23334500 D3331501 DARANE Dried blood body fluids and sperm samples on filter paper can be processed Purification times 5 Preps 50 Preps 200 Preps using the following method We recommend using OB Specimen Paper OSP 01 HiBind DNA Minicolumns 5 50 200 and OSP 02 for spotting blood as this unique filter paper disintegrates when incubated in aqueous buffers allowing for the efficient recovery of DNA This kit 2 ml Collection Tubes 15 150 600 can also be used for samples collected by using other specimen collection papers Buffer BL 5 ml 20 ml 60 ml 1 Cutor punch out the blood spot or other sample from the filter paper Up Buffer STL 5 ml 20 ml 50 ml Uer a m m to 200 ul of blood can be used for each spot Tear or cut filter into small Buffer HB 5 ml 30 ml 110 ml pieces and place into a microfuge tube DNA Wash Buffer 5 ml 20 ml 3x20 ml Note Use 3 4 punched cycles 3mm diameter for each DNA isolation Elution Buffer 2 mi 30 ml 2x50 ml OB Protease 3 mg 30 mg 4 x 30 mg 2 Add 200 ul Buffer STL and incubate at 55 C for 15 minutes Vortex every 2 User Manual 1 1 1 min to mix 3 Add 25 ul OB protease solution and mix by votexing Incubate for 45 minutes Note The E Z N A Forensic DNA Kit is supplied with enough buffer for the PP 9 at 60 C with occasional mixing Briefly centrifuge to remove any droplets standard protocol However due to increased volumes called for in some p
8. ntrate Dilute Wash Buffer with the indicated volume of absolute ethanol before use Washing Incomplete lysis Buffer BL is viscous and the leaves colored due to improper sample must be vortexed residue in mixing with Buffer thoroughly column BL 11 If the above suggestions fail to resolve any problems you are having with the E Z N A Forensic DNA Isolation Kit please feel free to contact our technical specialists at Tel 800 832 8896 toll free 770 931 8400 local international Fax 888 624 1688 toll free 770 931 0230 local international Or direct your questions via e mail to info omegabiotek com Orders can be placed via telephone facsimile or e mail NOTES
9. ollowing swab types cotton C E P Life Science Typical yields from these swabs are 0 5 3 ug 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 2 Separate the swab from the stick Place the buccal swab into a 2 0 mL microcentrifuge tube and add 550 ul PBS to the tube 3 Add 25 ul OB protease solution and 550 ul Buffer BL to the sample Mix immediately by votexing for 30 seconds Incubate 30 min at 60 C with occasional mixing Briefly centrifuge to remove any droplets from inside the lid 4 Add 550 ul absolute ethanol and mix thoroughly by vortexing Briefly centrifuge to remove any droplets from inside the lid 5 Insert the HiBind DNA Minicolumn into a 2 ml collection tube provided Carefully apply 600 ul of the mixture from Step 4 into the column Centrifuge at 8 000 xg for 1 min to bind DNA Discard flow through liquid and reuse the collection tube for the next step 6 Insert the column into a new 2 ml collection tube Carefully apply remaining volume about 500 ul of the mixture from Step 4 into the column Centrifuge at 8 000 x g for 1 min to bind DNA Discard the flow through liquid 7 Follow the standard E Z N A forensic DNA protocol from Step 7 on page 4 i e apply sample to the HiBind DNA spin column Note Incu
10. rotocols such as the buccal swab protocol fewer preparations may be from inside the lid 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 4 Add 225 ul Buffer BL and votex to mix Incubate at 60 C for 10 minutes information Briefly centrifuge to remove any droplets from inside the lid 5 Add 225 ul absolute ethanol and mix thoroughly by vortexing Briefly trif t droplets f inside the lid Before Starting centrifuge to remove any droplets from inside the li 6 Insert each HiBind DNA Minicolumn into a 2 ml collection tube provided Transfer the entire sample from Step 5 into the column including any precipitate that may have formed Centrifuge at 8 000 x g for 1 min to bind DNA Discard collection tube and flow through liquid IMPORTANT Reconstitute OB Protease in 150 ul Trial Kit or 1 5 ml 50 and 200 preps 10 mM Tris HCl pH 8 Vortex vial briefly prior to use DNA Wash Buffer Concentrate must be diluted with absolute ethanol 96 100 as follows 7 Place each column into a second 2 ml tube and wash by pipetting 500 ul D3591 00 A dd 20 mi absolute ethanol of Buffer HB into column Centrifuge at 8 000 x g for 1 min Dispose of flow f through liquid and re use the collection tube pas91 91 Add 80 ml absolute ethanol D3591 02 Add 80 ml absolute ethanol bottle 8 Place each column into a same 2 ml tube from step 7 and
11. wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol into column Centrifuge at 8 000 x g for 1 min Dispose of collection tube and flow through liquid All centrifugation steps must be performed at room temperature Note DNA Wash Buffer is provided as a concentrate and must be diluted 10 11 12 with absolute ethanol as indicated on the bottle label and Page 3 If refrigerated the diluted wash buffer must be brought to room temperature before use Refrigeration is NOT recommended Using a new collection tube wash the column a second time with 700 ul of DNA Wash Buffer and centrifuge as above Discard flow through and re use the collection tube Using the same 2 ml collection tube centrifuge at maximum speed gt 10 000 x g for 2 minutes to dry the column This step is critical for removal of residual ethanol that might otherwise interfere with downstream applications Place the column into anuclease free 1 5 ml microfuge tube and add 50 100 ul of Elution Buffer preheated to 70 C Allow the tube to sit for 3 minutes at room temperature To elute DNA from the column centrifuge at 8 000 x g for 1 min Repeat the elution with a second volume of 50 100 ul Elution Buffer Note Incubation at 70 C rather than at room temperature will give a modest increase in DNA yield per elution Alternatively use of the first eluate for second elution will increase DNA concentration Blood spots from finger pricks usuall
12. y contain no more than 50 ul blood and yield approximately 500 ng to 1 ug DNA This is sufficient for PCR analysis To obtain higher DNA concentrations elute with 50 ul preheated Elution Buffer or TE and repeat with the first eluate 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 Buffer RS before starting 20 mM Tris Cl pH 8 0 20 mM EDTA 200 mM NaCl 80 mM DTT 4 SDS DTT oxidizes quickly in aqueous solutions and should also be added just before use Store the DTT stock solution 1 M at 20 C 1 Add 1 100 ul of sperm toa 1 5 ml microcentrifuge tube Bring the volume up to 250 ul with Elution Buffer 2 Add 100 ul Buffer RS and 25 ul OB Protease Vortex to mix and incubate at 55 C in a shaking waterbath to effect complete lysis If no shaking waterbath is available vortex the sample every 20 30 minutes Lysis time depends on amount and type of tissue but is usually under 1 hours 3 Add 200 ul Buffer BL and 210 ul absolute ethanol to the sample and mix by vortexing 4 Follow the standard E Z N A forensic DNA protocol from Step 6 on page 4 i e apply sample to the HiBind DNA Mini column Protocol For Isolation of Genomic DNA From Buccal Swabs This protocol has been tested for the f
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