Contents Introduction
Contents
1. Sample too viscous Suggestions 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 If using more than 250 ul of blood increase volumes of OB Protease Proteinase K Buffer BL and isopropanol Pass aliquots of lysate through one column successively Divide sample into multiple tubes adjust volume to 250 ul with 10 mM Tris HCl Low DNA yield Clogged column See above Low A ratio 260 A 280 Poor elution Improper washing Buffy Coat used Extended centrifugation during elution step Poor cell lysis due to incomplete mixing with Buffer BL Hemoglobin remains on column 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 Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on page 5 before use With Buffy Coat samples use absolute ethanol rather than isopropanol in step 5 page 6 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 BL immediately and completely After application of sample to column wash once w2. Z N A Blood DNA method is ready for applications such as PCR Southern blotting and restriction digestion E Z N A Blood DNA Kit uses the reversible nucleic acid binding properties of HiBind matrix combined with the speed of mini column spin technology A specifically formulated buffer system allows genomic DNA 30 60 kb to bind to the matrix Samples are first lysed under denaturing conditions and then applied to the HiBind DNA spin columns to which 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 Storage and Stability All components of the E Z N A Blood DNA Kit should be stored at 22 C 25 C Proteinase K should be stored at 15 C 25 C Under these conditions DNA has successfully been purified and used for PCR after 24 months of storage 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 Store Buffer BL at room temperature Expiration Date All E Z N A Blood DNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22 25 C Binding Capacity Each HiBind column can bind approximately 30 ug DNA Using greater than 1 ml whole blood or 250 ul buffy coat is not recommended The PCR process is covered by U S Patents 4 683 195 and 4 683 202 and international equivalents o
3. for downstream application add 4 ul RNase A 50mg ml into the sample Add 25ul OB Protease and 500ul Buffer BL into sample Mix by vortexing at maxi speed for 30s Incubate at 65 C for 10 minutes Collect any liquid drop from lid by brief centrifugation Add 500ul absolute ethanol room temperature 96 100 to the sample and mix by vortexing at maxi speed for 15 seconds Collect any liquid drop from lid by brief centrifugation Carefully apply 750ul sample from step 4 into HiBind DNA Mini column pre inserted in a 2 ml collection tube provided Centrifuge at 8000 x g for 1 minute Discard the flow through and collection tube Place the column into a new 2 ml collection tube provided Carefully apply remaining sample from step 4 into HiBind DNA column Centrifuge at 8000 x g for 1 minute Discard the flow through and collection tube Place the column into a new 2 ml collection tube provided Add 500ul of HB Buffer into the column Centrifuge at 8000 x g for 1 minute Discard the flow through and reuse the collection tube in next step Place the column into a same 2 ml collection tube from step 7 Add 700ul of DNA Wash Buffer into the column Centrifuge at 8000 x g for 1 minute Discard Page 7 of 12 10 11 the flow through and reuse the collection tube in next step Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle and page 3 If refriger
4. step is crucial for ensuring optimal elution in the following step 10 Place the column into a sterile 1 5 ml microfuge tube and add 50 100 ul of preheated 65 C Elution Buffer Allow tubes to sit for 5 min at room temperature 11 To elute DNA from the column centrifuge at 8 000 x g for 1 min Retain flow through containing the DNA Discard column and store the eluted DNA at 20 C Blood spots from finger pricks usually contain no more than 50 ul blood and yield approximately 500 ng to 1 ug DNA This is usually sufficient for PCR analysis To obtain higher DNA concentrations elute with 50 ul preheated Elution Buffer or TE and repeat with the first eluate F Buffy Coat DNA Spin Protocol The buffy coat fraction of whole blood is enriched with WBC and usually gives at least 5 fold more DNA than the same volume of blood To prepare buffy coat from fresh whole blood simply centrifuge the sample at 3 000 4 000 x g for 10 min at room temperature Three layers should be obtained with plasma in the upper layer leucocytes in the middle layer buffy coat and erythrocytes in bottom layer Carefully aspirate the plasma making sure not to disturb the layer of concentrated leukocytes The buffy coat can be drawn off with a pipette and used directly in the E Z N A Blood DNA Protocol or frozen at 70 C for storage Page 10 of 12 Troubleshooting Guide Problem Clogged Column Possible Cause Incomplete lysis Sample too large
5. volume reduces the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 ul to 100 ul Elution Buffer Volumes lower than 50 ul greatly reduce yields Alternatively use the first eluate to perform the second elution If necessary the DNA can be concentrated Add sodium chloride to a final concentration of 0 1 M followed by 2 x volume of absolute 100 ethanol Mix well and incubate at 20 C for 10 min Centrifuge at 10 000 x g for 15 min and discard supernatant Add 700 ul of 80 ethanol and centrifuge at 10 000 x g for 2 min Discard supernatant air dry the pellet 2 min and resuspend DNA in 20 ul sterile deionized water or 10 mM Tris HCl pH 8 The expected yield from 250 ul blood is approximately 4 12 ug DNA Page 5 of 12 B Blood amp Body Fluid DNA Vacuum Spin Protocol Material and equipments supplied by user Tabletop microcentrifuge and sterile 1 5 ml tubes Vacuum Manifold Water bath set to 65 C m Ethanol approximately 0 3 ml per sample m RNase A Prepare a stock solution of RNase A at 50mg ml 1 Prepare the lysate by following step 1 5 of Protocol A Spin protocol on page 4 2 Insert the HiBind DNA Mini column into the vacuum manifold Carefully apply the lysate to an HiBind DNA column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Note If the lysate has difficulty to pass through the column at this stage Pl
6. BS and incubate at 65 C for 1 hour Vortex to mix every 20 min 3 Add 25 ul OB protease and mix well Incubate 30 min at 60 C with occasional mixing 4 Centrifuge at 10 000 x g for 5 min at room temperature Transfer the supernatant to a clean microfuge tube and add ONE volume of Buffer BL followed by ONE volume of absolute ehthanol Vortex thoroughly to mix at maxi speed for 30s Collect any drop from the lid by briefly centrifugation 5 Assemble an HiBind DNA column in a 2 ml collection tube provided Transfer the lysate from step 4 into the column and centrifuge at 8 000 x g for 1 min to bind DNA Discard the collection tube and flow through liquid 6 Place the column into a second 2 ml tube and wash by pipetting 500 ul of Buffer Page 9 of 12 HB Centrifuge at 8 000 x g for 1 min Again Discard flow through liquid and reuse the collection tube for next step 7 Place the column into a same 2 ml tube from step 7 and wash by pipetting 700 yl of DNA Wash Buffer diluted with ethanol Centrifuge at 8 000 x g for 1 min Again dispose of collection tube and flow through liquid 8 Using a new collection tube provided wash the column with a second 700 ul of DNA Wash Buffer diluted with ethanol and centrifuge as above Discard flow through and re use the collection tube for next step 9 Place the empty column into the same 2ml collection tube form step 8 centrifuge at maximum speed 15 000 x g for 2 min to dry the column This
7. Contents Introductio oe Sa i n Ana tp des Ha eb Se ee eat eee des SA 2 Storage and Stability 2 2 2 0 02 eee 2 Binding Capacity ici ssc eatin ote ee dae Pee bee ee a 2 Kit Contentsy ss ci24 ae ay deltas eee ce wee nea a Ga a aa a 3 Before Starting 42 os a Merk dee Mil ea lore ae Shh eh ok See Mia Nhe ee hg 3 A Blood amp Body fluid DNA Spin Protocol 2000000 eee aee 4 B Blood amp Body fluid DNA Vacuum Spin Protocol 2 00 5 C Buccal Swab DNA Spin Protocol 0 2 0 0 0 cee eee 7 D Buccal Swab DNA Vacuum Spin Protocol 00220000 8 E Dried Blood Samples Spin Protocol 20200 cee ee eeeee 9 F Buffy Coat DNA Spin Protocol 00 0000 cee eee 10 Troubleshooting Guide 00000 cee 11 Introduction E Z N A Blood DNA Kit provides a rapid and easy method for the isolation of genomic DNA from 1 pl 250 ul fresh frozen and anticoagulated whole blood The method can also be used for preparation of genomic DNA from buffy coat serum plasma saliva Buccal Swab and other body fuilds The kit allows single or multiple simultaneous processing of samples in under 30 minutes Normally up to 1 ml of whole blood can be used in a single experiment There is no need for phenol chloroform extractions and time consuming steps such as CsCl gradient ultracentrifugation and precipitation with isopropanol or ethanol are eliminated DNA purified using the E
8. ace the column into a collection tube supplied Close the lid and centrifuge at 8000 x g for 5 minutes or until all liquid pass through the column Place the column into another collection tube supplied and continue step 7 of the spin protocol 3 Pipet 500 ul of Buffer HB into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum 4 Wash the column by pipetting 750 ul of DNA Wash Buffer diluted with ethanol into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum 5 Close the lid of HiBind DNA column remove it from the vacuum manifold Insert the column into a collection tube supplied and centrifuge at 15 000 x g for 2 minute to completely dry the column 6 Elute DNA as Step 11 12 on page 5 Page 6 of 12 C Buccal Swab DNA Spin Protocol Important Note Due to the increased volume of Buffer BL that required for buccal swab protocol fewer preparation can be performed Additional BL Buffer can be purchased separately Product BL 100 All centrifugation steps are carried out at room temperature Material and Equipments required Tabletop microcentrifuge and sterile 1 5 ml tubes Water bath set to 65 C PBS Buffer Absolute Ethanol approximately 0 3 ml per sample RNase A Optional Prepare a stock of RNase A at 50mg ml Place the buccal swab in a 2 ml centrifuge tube Add 500 ul PBS in the tube If RNA free DNA is required
9. ated the diluted wash buffer must be brought to room temperature before use Place the column into a same 2 ml collection tube from step 8 Centrifuge at 15 000 x g for 2 minutes to completely dry the HiBind DNA Mini column Discard the flow through and the collection tube Place the column into a sterile 1 5 ml microfuge tube and add 50 100 ul of preheated 65 C Elution Buffer Allow tubes to sit for 5 min at room temperature To elute DNA from the column centrifuge at 8 000 x g for 1 min Retain flow through containing the DNA Place column into a second 1 5 ml tube Elute DNA again as step 10 11 Discard column and store the eluted DNA at 20 C D Buccal Swab DNA Vacuum Spin Protocol Material and Equipments required 4 Tabletop microcentrifuge and sterile 1 5 ml tubes Vacuum Manifold Water bath set to 65 C PBS Buffer Absolute Ethanol approximately 0 3 ml per sample RNase A Optional Prepare a stock of RNase A at 50 mg ml Prepare Buccal Swab lysate by following step 1 4 of Spin Protocol on page 7 Insert the HiBind DNA column into the vacuum manifold Carefully apply the lysate to an HiBind DNA column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Note If the lysate has difficulty to pass through the column at this stage Place the column into a collection tube supplied Close the lid and centrifuge at 8000 x g for 5 minutes or until all liquid pass through the
10. column Place the column into another collection tube supplied and continue step 7 of the spin protocol Apply remaining lysate into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Pipet 500 ul of Buffer HB into the column Turn on the vacuum source to draw Page 8 of 12 all liquid through the column Turn off the vacuum 5 Wash the column by pipetting 750 ul of DNA Wash Buffer diluted with ethanol into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum 6 Close the lid of HiBind DNA column remove it from the vacuum manifold Insert the column into a collection tube supplied and centrifuge at 15 000 x g for 2 minute to completely dry the column 7 Proceed Step 10 110f Baccul Swab DNA Spin Protocol on page 7 8 E Dried Blood Samples DNA Spin Protocol The following protocol is designed for isolating genomic DNA from dry blood sample collected with filter paper or commercial blood collecting card Material and equipments supples by user Tabletop microcentrifuge and sterile 1 5 ml tubes Water bath set to 65 C PBS Buffer Absolute Ethanol approximately 0 3 ml per sample RNase A Optional Prepare a stock of RNase A at 50 mg ml 1 Cutor punch out the blood spot from the filter paper Up to 200 ul blood can be used for each spot Tear or cut filter into small pieces and place into a microfuge tube 2 Add 250 ul P
11. his procedure For DNA extraction from tissue and mouse tail we suggest using the E Z N A Tissue DNA Kit product number D3495 and D3496 To isolate viral RNA from serum or other non cellular body fluids use E Z N A Viral RNA Kit Bring samples and OB Protease solution to room temperature and have a water bath equilibrated to 65 C Preheat an aliquot of Elution Buffer approximately 0 5 ml per sample at 65 C Carry out all centrifugation steps at room temperature 1 Add sample to a sterile microcentrifuge tube and bring the volume up to 250 ul with 10 mM Tris HCl PBS or Elution Buffer provided 2 Add 25 ul OB Protease and 250 ul of Buffer BL Vortex at maxi speed for 15s to mix thoroughly If RNA free genomic DNA is required add 5ul RNase A 50mg ml to each sample 3 Incubate sample at 65 C for 10 min 4 Briefly vortex the tube once during incubation 5 Add 260 ul of absolute ethanol room temperature 96 100 to lysate and vortex at maxi speed for 20s to mix thoroughly Briefly centrifuge the tube to collect any drops from the inside of the lid 6 Assemble an HiBind DNA Mini column in a 2 ml collection tube provided Transfer the lysate from step 5 into the column and centrifuge at 8 000 x g for 1 min to bind DNA Discard the collection tube and flow through liquid 7 Place the column into a second 2 ml tube provided and wash by pipetting 500 ul of Buffer HB Centrifuge at 8 000 x g for 1 min Again Discard f
12. ith 300 ul Buffer AL Page 11 of 12 Problem Possible Cause Suggestions No DNA eluted Poor cell lysis due to Mix thoroughly with Buffer BL prior to improper mixing with loading HiBind column Buffer BL No ethanol added to Dilute Wash Buffer with the indicated Wash Buffer volume of absolute ethanol before Concentrate use Absolute ethanol not Before applying sample to column an added to Buffer BL aliquot of Buffer BL ethanol must be added See protocol above Washing leaves Incomplete lysis due Buffer BL is viscous and the sample colored residue in to improper mixing with Buffer BL must be mixed thoroughly column No ethanol added to Wash Buffer Concentrate Dilute Wash Buffer with the indicated volume of absolute ethanol before use Eluted material has Sample volume too Reduce sample volume and follow red brown color large directions Hemoglobin remains After applying sample wash column on column once with 300 ul Buffer BL Page 12 of 12
13. low through liquid and reuse the collection tube for next step Page 4 of 12 10 11 12 Place the column into a same 2 ml tube from step 7 and wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol Centrifuge at 8 000 x g for 1 min Again dispose of collection tube and flow through liquid Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 3 If refrigerated the diluted wash buffer must be brought to room temperature before use Using a new collection tube wash the column with a second 700 ul of DNA Wash Buffer and centrifuge as above Discard flow through and re use the collection tube for next step Place the empty column into the same 2 ml collection tube form step 9 centrifuge at maximum speed 15 000 x g for 2 min to dry the column This step is crucial for ensuring optimal elution in the following step Place the column into a sterile 1 5 ml microfuge tube and add 100 200 ul of preheated 65 C Elution Buffer Allow tubes to sit for 5 min at room temperature To elute DNA from the column centrifuge at 8 000 x g for 1 min Retain flow through containing the DNA Place column into a second 1 5 ml tube Elute DNA again as step 11 12 Discard column and store the eluted DNA at 20 C Note Each elution typically yields 60 70 of the DNA bound to the column Thus two elution generally give gt 90 However increasing elution
14. wned by Hoffmann LaRoche Inc Page 2 of 12 Kit Contents Product No D3392 00 D3392 01 D3392 02 Purification times 5 Preps 50 Preps 200 Preps HiBind DNA Mini columns 5 50 200 2 ml Collection Tubes 15 150 600 Buffer BL 5 ml 20 ml 60 ml Buffer HB 5 ml 30 ml 110 ml DNA Wash Buffer 5 ml 20 ml 3x20 ml Elution Buffer 5 ml 40 ml 160 ml OB Protease 140 ul 14m 4x1 4ml User Manual 1 1 1 CAUTION Buffer BL contains a chaotropic salt Use gloves and protective eyeware when handling this solution gt Before Starting IMPORTANT DNA Wash Buffer must be diluted with absolute ethanol 96 100 as follows D3392 01 Add 80 ml absolute ethanol D3392 02 Add 80 ml absolute ethanol per bottle Store diluted DNA Wash Buffer at room temperature All centrifugation steps must be carried out at room temperature D3392 00 Add 20 ml absolute ethanol Page 3 of 12 A Blood amp Body Fluid DNA Spin Protocol Materials and equipments Supplied by User Tabletop microcentrifuge and sterile 1 5 ml tubes Water bath set to 65 C Ethanol approximately 0 3 ml per sample RNase A Prepare a stock solution of RNase A at 50 mg ml NOTE The procedure below has been optimized for use with FRESH or FROZEN blood samples 1 to 250 ul in volume Anticoagulated blood Saliva Serum Buffy Coat or other Body Fluid can also be used In addition lt 10 leukocytes or cultured cells may be used with t
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