Introduction Principle Storage and Stability
Contents
1. 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 SQL collection paper buffer Shake the tubes frequently Poor elution Repeat elution or increase elution volume Incubation of column at 70 C for 5 min with ER Buffer may increase yields Standard 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 Specimen Paper for spotting blood as this unique filter paper disintegrates when incubated in aqueous buffers allowing for the efficient recovery of DNA This kit can also be used for samples collected by using other specimen collection papers 1 Cut or punch out the blood spot or other sample from the 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 Note Use 3 4 punched cycles 3mm diameter for each DNA isolation 2 Add 200 ul Buffer QTL and incubate at 60 C for 15 minutes Vortex every 2 min to mix 3 Add 25 ul Protease K solution and mix by votexing Incubate for 45 minutes at 60 C with occasional mixing Briefly centrifuge to remove any droplets from inside the lid 4 Add 225 ul Buffer DL and votex to mix Briefly centrifuge to remove any2. Poor cell lysis due to incomplete mixing with Buffer DL Incomplete cell lysis or protein degradation due to insufficient incubation Samples are rich in protein Buffer PW2 Concentrate must be diluted with absolute 100 ethanol as specified before use Resin from the column may be present in eluate Avoid 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 Repeat the procedure this time making sere to vortex the sample with Buffer DL immediately and completely Increase incubation time with Buffer QTL and protease Ensure that no visible pieces of tissue remain After applying to column wash with 300 ul of a 1 1 mixture of Buffer DL and ethanol and then with Buffer PW2 11 Troubleshooting Guide Use the table below to find solutions to any problems you may have with the Solar Forensic DNA Isolation Kit Possible Cause Suggestions Clogged Column 10 Low DNA yield Incomplete lysis Extend incubation time of lysis with Buffer QTL and protease Add the correct volume of Buffer DL 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 Protease K or Proteinase K Buffer QTL Buffer DL and ethanol Pass aliquots of lysate through one column
3. droplets from inside the lid 5 Add 225 ul absolute ethanol and mix thoroughly by vortexing Briefly centrifuge to remove any droplets from inside the lid 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 10 11 12 Place each column into a second 2 ml tube and wash by pipetting 500 ul of Buffer PW1 into column Centrifuge at 8 000 x g for 1 min Dispose of flow through liquid and re use the collection tube Place each column into a same 2 ml tube from step 7 and wash by pipetting 700 ul of Buffer PW2 diluted with ethanol into column Centrifuge at 8 000 x g for 1 min Dispose of collection tube and flow through liquid Note Buffer PW2 is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle label If refrigerated the diluted Buffer PW2 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 Buffer PW2 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 critica for removal of residual ethanol that
4. might otherwise interfere with downstream applications Place the column into a nuclease free 1 5 ml microfuge tube and add 50 100 ul of ER 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 ER 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 usually contain no more than 50 ul blood and yield approximately 500 ng to 1 ug DNA This is sufficient for PCR analysis To obtain 7 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 8 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 ER Buffer as blank DNA concentration is calculated as DNA Absorbanceze6o x 0 05 gil x Dilution factor The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm A rat
5. 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 Protease K solution and 550 ul Buffer DL 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 x g 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 Solar forensic DNA protocol from Step 7 i e apply sample to the HiBind DNA spin column 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 Protocol for Isolation of Bacterial DNA From Biological Fluids 1 Pellet bacteria by centrifuging 10 minutes at 8 000rp
6. No DNA eluted Washing leaves colored residue in column 12 Poor cell lysis due to improper mixing with Buffer DL Poor cell and or protein lysis in Buffer QTL Absolute ethanol not added to Buffer DL No ethanol added to Buffer PW2 Concentrate Incomplete lysis due to improper mixing with Buffer DL No ethanol added to Buffer PW2 Concentrate Mix thoroughly with Buffer DL prior to loading HiBind column Tissue sample must be cut or minced into small pieces Increase incubation time at 65 C with Buffer QTL to ensure that tissue is completely lysed Before applying sample to column an aliquot of Buffer DL ethanol must be added See protocol above Dilute Buffer PW2 with the indicated volume of absolute ethanol before use Buffer DL is viscous and the sample must be votexed thoroughly Dilute Buffer PW2 with the indicated volume of absolute ethanol before use Introduction The Solar 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 precipitat
7. cool ambient conditions a precipitate may form in the Buffer DL In case of such an event heat the bottle at 37 C to dissolve the precipitate Store Buffer DL at room temperature Kit Contents Product Number DH194 00 DH194 01 DH194 02 Purification times 5 Preps 50 Preps 200 Preps HiBind DNA Mini columns 5 50 200 2 ml Collection Tubes 15 600 Buffer DL 60 ml Buffer QTL 50 ml Buffer PW1 110 ml Buffer PW2 Concentrate 3x20 ml ER Buffer 100 ml Protease Storage Buffer 7 mi Protease K 4x 30 mg User Manual 1 Note The Solar Forensic DNA Kit is supplied with enough buffers for the standard protocol However due to increased volumes called for in some protocols such as the buccal swab protocol fewer preparations may be performed Also additional buffers can be purchased separately from Solar Bio Tek See the Accessories section in the catalog or call customer service for price information Before Starting IMPORTANT 1 Reconstitute Protease K in 150 ul Trial Kit or 1 5 ml 50 and 200 preps Protease Storage Buffer Vortex vial briefly prior to use 2 Buffer PW2 Concentrate must be diluted with absolute ethanol 96 100 as follows DH194 00 Add 20 ml absolute ethanol DH194 01 Add 80 ml absolute ethanol DH194 02 Add 80 ml absolute ethanol bottle All centrifugation steps must be performed at room temperature Low A260 A280 ratio Improper washing Extended centrifugation during elution step
8. io of A260 A280 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 ug DNA with two elution each 200 ul 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 from Step 4 Forensic DNA Kit Vacuum Spin 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 Steps 1 5 2 Prepare the vacuum manifold according to manufacturer s instructions and connect the V Spin column to the manifold 3 Load the sample Buffer DL ethanol mixture into the column 4 Switch on vacuum source to draw the sample through the column then turn off the vacuum 5 Wash the column by adding 500 ul Buffer PW1 Draw Buffer PW1 through the column by turning on the vacuum source 6 Wash the column by adding 700 ul Buffer PW2 Draw the Buffer PW2 through the column by turning on the vacuum source Repeat this step with another 700 ul Buffer PW2 higher DNA concentrations elute with 50 ul preheated ER Buffer or TE and repeat with the first eluate Protocol for Isolation of Genomic DNA from Sperm This p
9. ion with isopropanol or ethanol are eliminated DNA purified using the Solar Forensic DNA method is ready for applications such as PCR Southern blotting and restriction digestion The Solar Forensic DNA Kit is specially designed to work with the OB Specimen Collection Paper 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 Solar 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 107 cells is not recommended Storage and Stability All components of the Solar Forensic DNA Kit except the Protease K can be stored at 22 C 25 C Once reconstituted in water Protease K must be stored at 20 C Under these conditions performance of all components of the kit is guaranteed at least 18 months Under
10. m 2 Resuspend bacterial pellet with 200 yl SQL buffer 3 Follow the standard protocol 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 SQL buffer 4 Follow the standard protocol 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 SQL buffer 3 Add 25 ul Protease K solution and mix by votexing Incubate at 55 C in a shaking waterbath to affect complete lysis If no shaking waterbath is
11. rotocol 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 HCl 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 to a 1 5 ml microcentrifuge tube Bring the volume up to 100 pl with ER Buffer 2 Add 100 ul Buffer RS and 25 pl Protease K Vortex to mix and incubate at 55 C in a shaking waterbath to affect 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 hour 3 Add 200 ul Buffer DL and 210 ul absolute ethanol to the sample and mix by vortexing 4 Follow the standard Solar forensic DNA protocol from Step 6 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 following 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
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