Contents Introduction
Contents
1. Yield and Size of Purified DNA DNA yield depends on the number of nucleated cell numbers presented in the sample Yields from whole blood may vary widely The following table shows the typical yields obtained from different samples The purified DNA size can be up to 200kb Species and Material Amount of Starting Typical Yield material Human Whole Blood 50 ul 0 3 0 6 ug Yield varies depending on the quantity of white 100 pl 1 5 pg blood cells present 200 ul 3 10 pg 300 ul 5 15 ug 500 ul 7 23 pg Mouse Whole Blood 50 ul 0 2 0 6 ug 100 pl 0 5 1 0 ug 200 ul 2 5 ug 300 ul 4 7 ug Cultured Cells 2 x 10 cells 10 15 ug Kit Contents DO714 50 DO714 250 D0714 2000 Product Number processed Blood Volumes 50 ml 250 ml 2000 ml Buffer NL 140 ml 4x 175 ml 22 x 260 ml Buffer XL 30 ml 150 ml 5 x 220 ml Buffer EB hydration buffer 50 ml 250 ml 10 x 220 ml OB Protease 300 ul 1 4 ml 5 5 ml User Manual 1 1 1 Page 3 of 16 Before Starting Prepare the Buffer XL OB Protease mixture For each 1 ml whole blood mix 500pl Buffer XL with 5yl OB IMPORTANT Protease This mixture should be prepare under 10 minutes without the sample OB Protease will lost the activities Storage of Blood Samples The procedure can use whole blood treated with EDTA heparin or citrate with either fresh or frozen condition Fresher blood yield better results For short term storage for up to 2 weeks it is recommended to collect2. samples in under 90 minutes There is no need for phenol chloroform extractions and time consuming steps such as CsCl gradient ultracentrifugation are eliminated DNA purified using the E Z N A SQ Blood DNA Kit Il method is ready for applications such as PCR Southern blotting and restriction digestion Principle E Z N A SQ Blood DNA Kit II uses a highly efficient solution based system to provide a convenient fast reliable and non toxic method to isolate high molecular weight genomic DNA from whole blood or buffy coat Plasma membrane are first lysed with Buffer NL cell nuclei and mitochondria are then pelleted by centrifugation The pellet is resuspended and lysed by Buffer XL which contains chaotropic salt and proteinase This step effectively removes most contaminant such as proteins High quality genomic DNA is then purified by isopropanol precipitation Storage and Stability All components of the E Z N A SQ Blood DNA Kit II should be stored at 22 25 C OB Protease should be stored at 15 25 C Under cool ambient conditions a precipitate may form in the Buffer XL In case of such an event heat the bottle at 55 C to dissolve Expiration Date All E Z N A SQ DNA Kit Il components are guaranteed for at least 24 months from the date of purchase when stored at 22 25 C The PCR process is covered by U S Patents 4 683 195 and 4 683 202 and international equivalents owned by Hoffmann LaRoche Inc Page 2 of 16
3. 5 minute at room temperature DNA will be OB Protease 1 5 ul 2 yl 2 5 ul visible as a small white pellet 100 isopropanol 150pl 200ul 250ul 10 Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 100 ul of 70 ethanol and vortex the tube for 10 seconds to wash the DNA pellet 70 ethanol 150ul 200ul 250ul Buffer EB 200ul 200ul 200uL 11 Centrifuge at 14 000 x g for 2 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and 1 Add 200 pl whole blood to a nuclease free 1 5 or 2 ml microcentrifuge tube watch the pellet containing 500 pl Buffer NL 2 5 x volume of blood Mix by inverting the tube 5 times 12 Invert the tube on a clean absorbent paper towel and air dry the pellet for 10 2 Centrifuge at 14 000 x g for 30 seconds at room temperature 15 minutes 13 Add 50 200 pl of DNA rehydration solution Buffer EB and vortex for 1 3 Discard the supernatant and leave the tube inverted on a clean piece of minute to mix absorbent paper for 2 min taking care that the pellet remains in the tube 4 Add 100 pl Buffer XL OB Protease 0 5 volume of blood mixture to the tube 14 Incubate sample at 65 C for 10 min Some samples may need to incubate at containing the nuclei pellet Vortex immediately for 10 seconds or until the 65 C for 1 hour to rehydrate DNA pellet is completely homogenized 15 Store DNA at 2 8 C For long term s
4. 7 5 ml 9 Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 1ml of 70 ethanol and vortex the tube for 10 seconds to wash the DNA pellet Buffer XL 0 5 ml 1ml 1 5 ml OB Protease 5 ul 10 ul 15 ul 100 isopropanol 0 5 ml 1 ml 1 5 ml 10 Centrifuge at 2000 x g for 3 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and 70 ethanol 0 5 ml 1 ml 1 5 ml watch the pellet Buffer EB 0 2 ml 0 2 ml 0 3 ml 11 Invert the tube on a clean absorbent paper towel and air dry the pellet for 10 1 Add 2 ml whole blood or bone marrow to a nuclease free 15 ml centrifuge 15 minutes tube containing 5 ml Buffer NL 2 5 volume of blood Mix by inverting the tube 5 times 12 Add 200 500 ul of DNA rehydration solution Buffer EB 2 Centrifuge at 2 000 x g for 5 minutes at room temperature 13 Incubate sample at 65 C for 10 min Some sample may need to incubate at 65 C for 1 hour to rehydrate DNA 3 Discard the supernatant and leave the tube inverted on a clean piece of absorbent paper for 2 min taking care that the pellet remains in the tube 14 Store DNA at 2 8 C For long term storage store at 20 C 4 Add 1 ml Buffer XL OB Protease mixture 0 5 x volume of blood to the tube containing the nuclei pellet Vortex immediately for 10 seconds or until the pellet is completely homogenized Tips OB Protease and Buffer XL can be premix tog
5. minutes without the samples OB Protease will lose activities Important When process multiple samples vortex each tube immediately after addition of XL OB Protease mixture Page 12 of 16 5 Centrifuge at 10 000 x for 5 seconds to collect any liquid drop from lid 6 Incubate at 65 C for 10 30 minutes in a water bath or heating block 7 Add 200 ul isopropanol to the lysate 8 Gently mix the solution by inverting the tube 20 30 times or until the DNA precipitate become visible as threads or clumps 9 Centrifuge at 14 000 x g for 2 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and watch the pellet 10 Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 200pl of 70 ethanol and vortex the tube for 10 seconds to wash the DNA pellet 11 Invert the tube on a clean absorbent paper towel and air dry the pellet for 10 15 minutes 12 Add 200 ul of DNA rehydration solution Buffer EB Incubate sample at 65 C for 10 min Some samples may need to incubate at 65 C for 1 hour to rehydrate DNA 13 Store DNA at 2 8 C For long term storage store at 20 C F DNA Purification Protocol for 1 2 x 10 Cultured Cells This protocol is designed for isolating genomic DNA from 1 2 million cultured cells 1 Harvest the cells and transfer them with salt balanced buffer such as PBS to a 2 0 ml tube For adherent cells trypsinize
6. the cells before harvesting 2 Centrifuge at 300 x g for 5 minutes to pellet the cells Remove and discard supernatant Leave the tube inverted on a absorbent paper for 2 3 minutes 3 Add 300 ul of Buffer NL and mix by pipetting up and down until the pellet is resuspended The lysate should be cloudy 4 Add 300 ul Buffer XL OB Protease mixture Vortex immediately for 10 Page 13 of 16 10 11 12 13 G seconds Incubate at 65 C for 10 30 minutes in a water bath or heating block Add 600 ul isopropanol to the lysate Gently mix the solution by inverting the tube 20 30 times or until the DNA precipitate become visible as threads or clumps Centrifuge at 14 000 x g for 2 minutes at room temperature Carefully pour off the ethanol Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 600ul of 70 ethanol and invert the tube a few times to wash the DNA pellet Centrifuge at 14 000 x g for 2 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and watch the pellet Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10 15 minutes Add 100 200 ul of DNA rehydration solution Buffer EB Incubate sample at 65 C for 10 min Some samples may need to incubate at 65 C for 1 hour to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C DNA Purification from up to 10 m
7. 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 SQ DNArotocol or frozen at 70 C for storage Reagent volumes required for processing 100 500pl Buffy coat Reagent Buffy Coat Volume 100pt 200 300pl 400pl 500ul Buffer NL 250ul 500ul 750ult 1000p 1250ul Buffer XL 100pl 200 300ul 400ul 500ul OB Protease 1ul 2 ul 3 ul 4ul 5 ul 100 isopropanol 100pl 200 300ul 400ul 500ul 70 ethanol 100pl 200 300ul 400ul 500ul Buffer EB 200ul 2200p 200ul 200ul 200ul 1 Add 200 ul buffy coat preparation to a nuclease free 1 5 ml microcentrifuge tube containing 500pl Buffer NL Mix by inverting the tube 5 times 2 Centrifuge at 14 000 x g for 30 seconds at room temperature 3 Remove and discard supernatant Leave the tube inverted on a absorbent paper for 2 minutes Make sure the pellet remains in the tube 4 Add 200ul Buffer XL OB Protease mixture to the tube containing the nuclei pellet Vortex immediately for 10 seconds or until the pellet is completely homogenized Tips OB Protease and Buffer XL can be premix togather Add 50ul OB Protease to 5 ml Buffer XL This Mixture should be prepared in 10
8. Contents Introductions wees So ete OR a etre Sa ea Ee ate a ge ela Be ae 2 Principle riesa Seek 8 Shey e au Fa Bees es Se oe ae ee E E 2 Storage and Stability 2 ee ee ees 2 Yield from Various Samples 0 ee eee ee eee eee eee 3 Kit GOntents s s6 beet eee he gr ge ka ee ene ae a ee a eee a ie 3 Before Startna es aston ea Sate oan Wed RIN Gib hoe eS 4 A Protocol for 100 500 pl whole blood 2 00 eee eee 5 B Protocol for 1 3 ml whole blood 2 02 e eee eee eee 7 C Protocol for 4 14 ml whole blood 2 2 ee ee ee ee ees 9 D Protocol for 20 ml whole blood 0 02 2 eee eee 11 E Protocol for 100 500 pl Buffy coat 2 2 2 2 ee ee ee eee 12 F Protocol for Cultured cells 2 2 ee ee ee ee ee eee 14 G Protocol for Clotted Blood ee ee ee ee ee ee ee 15 Troubleshooting Guide 2 eee ee ee ees 16 Introduction The E Z N A SQ Blood DNA Kit II is designed for isolating high molecular weight genomic DNA from fresh frozen and anticoagulated whole blood The method can also be used for preparation of genomic DNA from buffy coat bone marrow or cultured cells The procedure can be easily scaled up and down allowing purification from different amount of starting material The whole procedure can be performed in single tube so it can reduce the chance for potential cross contamination This kit allows single or multiple simultaneous processing of
9. ather Add 10ul OB Protease to 1000 ul Buffer XL This Mixture should be prepared in 10 minutes without the samples OB Protease will lose activities Important When process multiple samples vortex each tube immediately after addition of XL OB Protease mixture C DNA Purification Protocol for 4 14 ml whole blood NOTE The buffer volume of the following protocol is for isolating 12 ml whole blood 5 Incubate at 65 C for 10 30 minutes in a water bath or heating block sample This procedures can be scaled up and down for use with FRESH or FROZEN blood samples 4 ml to 14 ml in volume by scaling reagent volume up and down in Note The sample should change color from red to Oliver green during proportion to the volume of sample used Except the Buffer EB volume for 3 ml proteinase digestion blood Frozen blood should be thawed quickly in a 37 C water bath with gently agitation and stored on ice before starting the procedure 6 Add 1 ml isopropanol 0 5 volume of blood to the lysate Page 7 of 16 Page 8 of 16 Reagent volumes required for processing 4 14 ml whole blood samples Reagent Blood Volume 6 ml Buffer NL 15 ml Buffer XL 3 ml OB Protease 30 ul 100 isopropanol 3 ml 70 ethanol 3 ml Buffer EB 200ul Reagent Blood Volume 9 ml 10 ml 11 ml 12 ml 13ml 14ml Buffer NL 22 5 ml 25 ml 27 5 ml 30ml 32 5ml 35ml Buffer XL 4 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml OB Protease 45 ul 50 ul 50 ul 50 ul 50 ul 50 ul n
10. blood in the tube contains EDTA as anticoagulant For long term storage sample should be collected in the tube contains EDTA as anticoagulant and store at 70 C Materials to be supplied by user e Microcentrifuge capable of 14 000 x g Nuclease free 1 5 ml or 2 ml microcentrifuge tubes for 100 500 ul blood Nuclease free 15 ml microcentrifuge tubes for 0 5 3 ml blood Nuclease free 50 ml microcentrifuge tubes for 3 10 ml blood Water Bath preset at 37 C and 65 C 100 Isopropanol 70 ethanol A DNA Purification Protocol for 100 500ul whole blood NOTE The buffer volume of the following protocol is for isolating 200ul whole blood sample This procedures can be scaled up and down for use with FRESH or FROZEN blood samples 100 ul to 500 pl in volume by scaling reagent volume up and down in proportion to the volume of sample used Except the Buffer EB volume for 100pl blood Frozen blood should be thawed quickly in a 37 C water bath with gently agitation and stored on ice before starting the procedure Page 4 of 16 Reagent volumes required for processing 100 500pl whole blood samples proteinase digestion Reagent Blood Volume 7 Add 100 ul isopropanol 0 5 volume of blood to the lysate 300pt 4400p 500pl 8 Gently mix the solution by inverting the tube 20 30 times or until the DNA Buffer NL 750ul 1000p 1250pl precipitate become visible as threads or clumps Buffer XL 150ul 200 250uL 9 Centrifuge at 14 000 x g for
11. ease and Buffer XL can be premix togather Add 50ul OB Protease to 5 ml Buffer XL This Mixture should be prepared in 10 minutes without the samples OB Protease will lose activities Important When process multiple samples vortex each tube immediately after addition of XL OB Protease mixture Incubate at 65 C for 15 30 minutes in a water bath or heating block Add 5 ml isopropanol to the lysate Gently mix by inverting the tube 20 30 times or until the DNA precipitate become visible as threads or clumps Centrifuge at 2000 x g for 5 minute at room temperature Pour out the supernatant and add 5 ml of 70 ethanol and vortex the tube for 10 seconds to wash the DNA pellet Centrifuge at 2000 x g for 3 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and watch the pellet Invert the tube on a clean absorbent paper towel and air dry the pellet for 10 15 minutes Add 1 ml of DNA rehydration solution Buffer EB Incubate sample at 65 C for 10 min Some sample may need to incubate at 65 C for 1 hour to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C E DNA Purification Protocol for 100 500 pl Buffy Coat Page 11 of 16 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
12. ee 4 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 70 ethanol 4 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml Buffer EB 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 Add 12 ml whole blood or bone marrow to a nuclease free 50 ml centrifuge tube containing 30 ml 2 5 x volume of blood Buffer NL Mix by inverting the tube 5 times 2 Centrifuge at 2000 x g for 5 minutes at room temperature 3 Discard the supernatant and leave the tube inverted on a clean piece of absorbent paper for 2 min taking care that the pellet remains in the tube 4 Add 5 ml Buffer XL OB Protease mixture to the tube containing the nuclei pellet Vortex immediately for 10 seconds or until the pellet is completely homogenized Page 9 of 16 Tips OB Protease and Buffer XL can be premix togather Add 50ul OB Protease to 5 ml Buffer XL This Mixture should be prepared in 10 minutes without the samples OB Protease will lose activities Important When process multiple samples vortex each tube immediately after addition of XL OB Protease mixture 5 Incubate at 65 C for 15 30 minutes in a water bath or heating block Note The sample should change color from red to oliver green during proteinase digestion 6 Add 5 ml isopropanol to the lysate Gently mix by inverting the tube 20 30 times or until the DNA precipitate become visible as threads or clumps 7 Centrifuge at 2000 x g for 5 minute at room temperature DNA will be visible as a small white pellet 8 Pour out the supernatant and drai
13. inverting the tube 20 30 times Centrifuge at 2000 x g for 5 minutes to pellet the DNA Discard the supernatant and add 5 ml 70 ethanol and vortex for 10 seconds Centrifuge at 2000 x g for 5 minutes Discard the supernatant and invert the tube onto a clean absorbent paper for 10 minutes Add 1 ml Buffer EB or TE Buffer vortex 5 seconds at lower speed Dissolve the DNA by incubating 1 hour at 65 C or overnight at room temperature Page 15 of 16 Troubleshooting Guide Low DNA Blood Sample contains too Draw new blood samples yield few white blood cells Blood sample is too old Try to use fresh blood if possible Buffer XL OB Protease is not prepared correctly Ensure that the Buffer XL OB Protease is not prepared correctly OB Protease is dissolved in Use EB to dissolve the proteinase wrong buffer Incomplete sample lysis Mix the sample throughly after addition of Buffer NL DNA pellet was lost during Be very careful not to lose the DNA isopropanol precipitation when removing isopropanol or ethanol during precipitation and wash steps Proteinase digestion was not complete make sure to prepare the XL OB Protease properly and fresh Poor cell lysis due to incomplete mixing with Buffer NL Repeat the procedure this time making sure to vortex the sample with Buffer NL immediately and completely Hemoglobin remains Repeat the procedure this time making sure enough volume of NL b
14. l Clotted Blood Transfer 10 ml the clotted blood including any liquid residual into a 50 ml centrifuge tube Homogenize the sample with a rotor stator homogenizer until the sample is uniformly homogenous Add 25 ml Buffer NL and mix by inverting the tube 5 7 times Centrifuge at 2000 x g for 5 minutes in a swing out rotor Discard the supernatant and add another 10 ml Buffer NL to the pellet Vortex to resupsend the pellet Centrifuge at 2000 x g for 5 minutes in a swing out rotor Page 14 of 16 10 11 Discard the supernatant and add leave the tube inverted on a clean absorbent paper for 2 minutes Make sure that the pellet remain in the tube Add 5ml Buffer XL and 50 pl OB Protease solution 20mg ml close the cap and vortex immediately until the pellet is completely homogenized Note When processing multiple samples vortex each tube immediately after addition of Buffer XL OB Protease Although the pellet can be easily homogenized with few pulses of high speed vortexing however traces of pellet with a jelly like consistency often barely visible may remain If these traces are seen vortex sample for another 30 seconds Incubate the tube at 65 C for 30 minutes in a water bath or heating block Vortex for 10 seconds inspect the tube to make sure the homogenization is complete Centrifuge at 2000xg for 5 to remove undigested particles Transfer the supernatant into a new tube Add 5 ml of isopropanol and mix throughly by
15. n the tube briefly on a clean absorbent paper towel Add 5 ml 70 ethanol and vortex the tube for 10 seconds to wash 9 Centrifuge at 2000 x g for 3 minutes at room temperature Carefully pour off the ethanol Pellet may be very loose at this point so pour slowly and watch the pellet 10 Invert the tube on a clean absorbent paper towel and air dry the pellet for 10 15 minutes 11 Add 1 ml of DNA rehydration solution Buffer EB 12 Incubate sample at 65 C for 10 min Some sample may need to incubate at 65 C for 1 hour to rehydrate DNA 13 Store DNA at 2 8 C For long term storage store at 20 C D DNA Purification Protocol for 20 ml whole blood 1 Add 10 ml whole blood to a nuclease free 50 ml centrifuge tube containing 25 ml Buffer NL Mix by inverting the tube 5 times 2 Centrifuge at 2000 x g for 5 minutes at room temperature Remove and discard supernatant Page 10 of 16 10 11 12 13 14 Pipet 25 ml Buffer FG1 into the same 50 ml centrifuge tube Add another 10 ml whole blood and mix by inverting the tube 5 times Centrifuge at 2000 x g for 5 minutes at room temperature Discard the supernatant and leave the tube inverted on a clean piece of absorbent paper for 2 min taking care that the pellet remains in the tube Add 5 ml Buffer XL OB Protease mixture to the tube containing the nuclei pellet Vortex immediately for 10 seconds or until the pellet is completely homogenized Tips OB Prot
16. torage store at 20 C Tips OB Protease and Buffer XL can be premix togather Add 1ul OB Protease to 100 ul Buffer XL This Mixture should be prepared in 10 minutes without the samples OB Protease will lose activity Important When process multiple samples vortex each tube immediately after addition of XL OB Protease mixture B DNA Purification Protocol for 1 3 ml whole blood NOTE The buffer volume of the following protocol is for isolating 2 ml whole blood sample This procedures can be scaled up and down for use with FRESH or FROZEN blood samples 1 ml to 3 ml in volume by scaling reagent volume up and down in proportion to the volume of sample used Except the Buffer EB volume for 3 ml blood Frozen blood should be thawed quickly in a 37 C water bath with gently agitation and stored on ice before starting the procedure 5 Centrifuge at 10 000 x for 5 seconds to bring down any liquid drop from tube lid 6 Incubate at 65 C for 10 30 minutes in a water bath or heating block Note The sample should change color from red to oliver green during Page 5 of 16 Page 6 of 16 Reagent volumes required for processing 1 3 ml whole blood samples 7 Gently mix the solution by inverting the tube 20 30 times or until the DNA precipitate become visible as threads or clumps Reagent Blood Volume 8 Centrifuge at 2000 x g for 5 minute at room temperature DNA will be visible 1 ml 2 ml 3 ml as a small white pellet Buffer NL 2 5 ml 5 ml
17. ufefr is used DNA pellet was lost during Be very careful not to lose the DNA isopropanol precipitation when removing isopropanol or ethanol during precipitation and wash steps DNA Pellet is DNA pellet was over dried Rehydrate the DNA by incubating the difficult to DNA pellet with Buffer EB at 65 C for 1 dissolve hour and then leave the sample at room temperature or 4 C for overnight DNA pellet was not mixed well during rehydration step Shake a few times during the rehydration step Gel like traces After addition of the XL OB Immediately mix the sample after the addition of XL OB Protease of pellet Protease the sample was remaining after resuspension of pellet in XL Proteinse mixture left too long before the vortexing Page 16 of 16
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