Manual - Omega Bio-Tek
Contents
1. Optional Add 3 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes 7 Cool the sample to room temperature 8 Add 200uL PCP Buffer to the cell lysate 9 Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes 10 Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 11 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 600 uL of 100 isopropanol 12 Gently mix the solution by inverting the tube 30 40 times 13 Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet 14 15 16 17 18 19 Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 600 uL 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 uL of DNA rehydration solution Buffer EB and vortex for 1 minutes to mix Incubate samp2. The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 20 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 1 35 mL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The 10 11 12 13 14 15 16 17 18 precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 4 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 4 mL of 70 ethanol and invert the tube few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out
3. be diluted with ddH O according to bottle label before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 350 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely resuspended Add 12 mL WTL Buffer to the tube containing the resuspended cells Vortex for 2 minutes to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 50 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 4 0 mL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing 10 11 12 13 14 15 16 17 18 Centrifuge at 2 000 x g or higher for 10 minutes at room temperature The precipitated prote
4. pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 1 5 mL centrifuge tube containing 200uL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 200uL 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 65 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix 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 Page 8 of 45 C DNA Purification Protocol for 300uL whole blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 1 5 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C lsopropanol 70 ethanol Add 300uL whole blood or bone marrow to a nuclease free 1 5 mL microcentrifuge tube containing 900uL ERL Buffer Mix by inverting the tube a fe
5. room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 100 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely resuspended Add 1 mL WTL Buffer to the tube containing the resuspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 5 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 335 uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The 10 11 12 13 14 15 16 17 18 precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tu
6. seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at 2000 x g for 10 minutes at room temperature The precipitated protein will form a tight pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 6000 uL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 2000 x g for 10 minutes at room temperature DNA will be visible as a small white pellet 14 15 16 17 18 19 Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 6000 uL of 70 ethanol and invert the tube a few times to wash the DNA pellet Centrifuge at 2000 x g for 10 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 1000 uL of DNA rehydration solution Buffer EB and vortex for 2 minutes to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 36 of 45 R DNA Purification from 50uL Clotted Blood 17 Incubate sample at 65 C for 1 hour
7. the pellet Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 400 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and overnight at room temperature to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 22 of 45 J DNA Purification Protocol for 5 mL Whole Blood Materials to be supplied by user Centrifuge capable of 2 000 xg Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 5 mL whole blood or bone marrow to a nuclease free 15 mL microcentrifuge tube containing 15 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 350 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ER
8. 37 C for approximately 10 minutes Cool the sample to room temperature Add 1 mL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Centrifuge at 2 000 x g for 5 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 10 11 12 13 14 15 16 17 18 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 3 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 3 mL of 70 ethanol and invert the tube a few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out the pellet Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 250 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubat
9. Contents INtOdUCTION 22 c4 E lee ER tas bank net aia Maio ie A as 2 Principle tse saute Aico eerie eae i eh 2 Storage and Stability 0 0 0 00 0000 cee 2 DNA Yield from Various Samples 000 000s 4 Kit Contents 0 sec ant sha ree eka Ebates Dont shee of Ae Sag 3 Before Starting enre 20 be et hee eae Ve Ee Se 3 A Protocol for 100 uL whole blood 000000 eee eee 5 B Protocol for 200 uL whole blood 2020 00 e eee eee 7 C Protocol for 300 uL whole blood 000 0c 9 D Protocol for 600 uL whole blood 002000 cece eee 11 E Protocol for 800 uL whole blood 20000 0 eee eee eee 13 F Protocol for 1 mL whole blood 000220 cece eee eee 15 G Protocol for 2 mL whole blood 2 0000 cece eee 17 H Protocol for 3 mL whole blood 000 02 ee eee eee 19 I Protocol for 4 mL whole blood 000 0 eee eee eee 21 J Protocol for 5 mL whole blood 00020 cee eee eee eee 23 K Protocol for 6mL whole blood 00 00 e eee eee eee 25 L Protocol for 12 whole blood 00 00 e eee 27 M Protocol for Buffy coat 1 2 2 0 0 0 0 0 cee 29 N Protocol for Cultured cells 0 5 1x10 0 0 0 0 ccc cee eee 31 O Protocol for Cultured cells 3 5 x 10 n n nan cee ee 33 P Protocol for Cultured cells 3 5 X10 0 ene 35 Q Protocol for 50 uL Clotted Blood 0 ee
10. L Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended Add 5 mL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 25 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 1 70 mL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing 10 11 12 13 14 15 16 17 18 Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 5 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube b
11. age 4 above before use Centrifuge at 14 000 x g for 30 seconds at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 15uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended Add 200uL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 1 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes Cool the sample to room temperature Add 67uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at max speed for 3 minutes at room temperature The 10 11 12 13 14 15 16 17 18 precipitated protein will form a tight dark brown
12. and room temperature for overnight to 1 Transfer the 50uL blood include any liquid residual into a 1 5 mL centrifuge rehydrate DNA Gently shake tube several times during incubation to tube disperse DNA 2 Add 550uL WTL Buffer and pipet up an down few times to mix 18 Store DNA at 2 8 C For long term storage store at 20 C 3 Add 3 uL Proeinase K solution 25mg mL and mix by inverting 20 times 4 Incubate at 55 C for 1 hour to overnight until clots has dissolved 5 Place the tube on ice for 1 minute 6 Add 3 uL RNase A to the cell lysate and invert 10 time to mix throughly Incubate the tube at 37 C for 5 minutes 7 Place the tube on ice for 1 minute 8 Add 200 uL PCP buffer to the cell lysate 9 Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes 10 Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat this step 11 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 600uL of 100 isopropanol If the DNA yield is expected to be lower than 2ug add 2uL of glycogen 20mg mL per sample 12 Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet 13 Pour of t
13. and watch the pellet Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10 15 minutes Add 100 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 10 min Some samples may need to incubate at 65 C for 1 2 hour to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C Page 12 of 45 E DNA Purification Protocol for 800 uL whole blood Materials to be supplied by user 19 20 21 22 23 24 25 26 Centrifuge capable of 2 000 x g Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 800 uL whole blood or bone marrow to a nuclease free 15 mL centrifuge tube containing 2 4 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during the incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 50 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resus
14. be in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 1 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 1 mL of 70 ethanol and invert the tube a few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out the pellet Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10 15 minutes Add 100 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 2 hour to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Some samples may need to incubate at room temperature overnight to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C Page 16 of 45 G DNA Purification Protocol for 2 mL Whole Blood Materials to be supplied by user Centrifuge capable of 2 000 x g Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 2 mL whole blood or bone marrow to a nuclease free 15 mL microcentrif
15. cell clumps visible at this point 5 Add 150uL of WTL Buffer to the resuspended cells and mix by pipetting The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps are cannot be seen 6 Optional Add 1 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes 7 Cool the sample to room temperature 10 11 12 13 14 15 16 17 18 19 Add 50uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 150uL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 150uL 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 b
16. d on a clean absorbent paper for 2 minutes Make sure that the pellet remain in the tube 7 Add 5mL WTL Buffer and 50 uL Proteinase K solution 25mg 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 WTL Buffer Proteinase K Do not wait until buffer has been added to all samples before vortexing Although the pellet can be easily homogenized with 3 4 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 8 Incubate the tube at 65 C for 30 minutes in a water bath or heating block 9 After the lysate cool to room temperature add 1 7 mL PCP buffer Mix by vortexing for 15 seconds 10 Incubate at ice for 10 minutes 11 Centrifuge at 2000 4000 x g for 10 minutes to pellet the protein 12 Transfer the supernatant to a new 50 mL tube add 4 75mL of isopropanol and mix gently by invert the tube 20 30 times DNA precipitate will become visible as threads or a clump 13 Centrifuge at 2000 4000 x g for 10 minutes to pellet the DNA 14 Discard the supernatant and briefly invert the tube onto a clean absorbent paper make sure the DNA pellet remains inside the tube 15 Add 5 mL 70 ethanol and vortex for 10 seconds 16 Centrifuge at 2000 4000 x g for 5 10 minutes Transfer the clotted blo
17. e blood cells present 3 mL 50 150 ug Buffy Coat 5 15 ug Mouse Whole Blood 0 2 0 6 ug Cultured Cells 10 15 ug Guideline for Sample and Volume of Reagents The SQ Blood DNA system is a solution based system and the protocol can be easily modified based on the sample volume User can change the protocol based on the following guideline Page 4 of 45 Sample volume Reagent Name Reagent volume ERL Buffer WTL Buffer PCP Buffer 0 33 x Isopropanol 70 ethanol 1x EB Buffer 0 35 x _ SSS For lt 300ul blood volume Volume of EB Buffer used can be adjusted depends on the desired final concentration A DNA Purification Protocol for 100uL whole blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 1 5 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C lsopropanol 70 ethanol Add 100uL whole blood or bone marrow to a nuclease free 1 5 mL microcentrifuge tube containing 300uL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during the incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 14 000 x g for 30 seconds at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 10uL of residue
18. e eee ee 37 R Protocol for 1mL Clotted Blood 000 00 c eee 39 S Protocol for Large volume of Clotted Blood gt 1mL 41 Determination of Yield and Quality n a anana aaa 43 Troubleshooting Guide nannaa aeaa 44 Revised April 2008 Introduction The E Z N A SQ Blood DNA Kit is designed for isolating high molecular weight genomic DNA from fresh frozen and anti coagulated whole blood The method can also be used for preparation of genomic DNA from buffy coat bone marrow or cultured cells The kit allows single or multiple simultaneous processing of 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 method is ready for applications such as PCR Southern blotting and restriction digestion Principle E Z N A SQ Blood DNA Kit 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 Red blood cells are first lysed with ERL buffer followed by lysis of the white blood cells and their nuclei in the WTL Buffer Cellular proteins are removed by precipitation and high molecular weight genomic DNA will remain in solution High quality genomic DNA is then purified by isopropanol precipitation Storage and Stab
19. e tubes Water Bath preset at 37 C and 65 C lsopropanol 70 ethanol Add 600uL whole blood or bone marrow to a nuclease free 2 0 mL microcentrifuge tube containing 1 2mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 14 000 x g for 30 seconds at room temperature Remove and discard supernatant containing lysed red blood cells Leave behind any supernatant that appears to contain non lysed red blood cells Vortex the tube vigorously until the cell pellet is completely resuspended Add 1 2 mL ERL buffer to the tube and mix well by inverting the tube a few times Incubate at room temperature for 2 minutes Centrifuge at 14000 x g for 30 seconds at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 15 20uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 5 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Add 600uL WTL Buffer to the tube containing the
20. e 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 25 uL of DNA rehydration solution Buffer EB and vortex for 1 minutes to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 32 of 45 P DNA Purification Protocol for Cultured Cells 3 5 x 10 Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 2 0 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C Isopropanol 70 ethanol 1 This protocol is designed for isolating genomic DNA from 3 5 x 10 cultured cells 2 Harvest the cells and transfer them with salt balanced buffer such as PBS to a 2 0 mL microcentrifuge tube For adherent cells trypsinize the cells before harvesting 3 Centrifuge at 14 000 x g for 10 seconds to pellet the cells Remove the cells and leave behind about 25 uL residue liquid 4 Vortex the cells to resuspend the cells in the residue liquid Make no cell clumps visible at this point 5 Add 600 uL of WTL Buffer to the re suspended cells and mix by pipetting The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps are cannot be seen 6
21. ed for 30 seconds to mix Some protein clumps may be visible after vortexing Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not 10 11 12 13 14 15 16 17 18 tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 2 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 2 mL of 70 ethanol and invert the tube few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out the pellet Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 200 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 18 of 45 H DNA Purification Protocol fo
22. ge 44 of 45 Page 45 of 45
23. he 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 14 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 15 Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10 15 minutes 16 Add 20 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix Page 38 of 45 S DNA Purification from 1mL Clotted Blood rehydrate DNA Gently shake tube several times during incubation to 1 Transfer 1 mL clotted blood include any liquid residual into a 50 mL disperse DNA centrifuge tube 18 Store DNA at 2 8 C For long term storage store at 20 C 2 Add 11 mL WTL Buffer and pipet up an down few times to mix 3 Add 50 uL Proteinase K solution 25mg mL and mix by inverting 20 times 4 Incubate at 55 C for 3 hour to overnight until clots has dissolved 5 Place the tube on ice for 1 2 minute 6 Add 50 uL RNase A to the cell lysate and invert 10 time to mix throughly Incubate the tube at 37 C for 5 minutes 7 Place the tube on ice for 1 2 minute 8 Add 4 mL PCP buffer to the cell lysate 9 Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 10 minutes 10 Centrifuge at 2000 x g for 10 minutes at room temperature The
24. ility All components of the E Z N A SQ Blood DNA Kit should be stored at 22 C 25 C Under cool ambient conditions a precipitate may form in the Buffer WTL In case of such an event heat the bottle at 55 C to dissolve Expiration Date All E Z N A SQ Blood DNA Kit 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 45 Kit Contents Product D5032 00 D5032 01 D5032 02 D5032 03 l Total Blood Volume 10 mL 50 mL 150 mL 300 mL ERL Buffer 10 x 2x 80 mL Buffer WTL 10 mL 50 mL 150 mL 300 mL PCP Buffer 4mL 20 mL 60 mL 120 mL Before Starting ERL Buffer Concentrate must be diluted with ddH20 as follows before use IMPORTANT D5032 00 Add 45 mL water bottle D5032 01 Add 225 mL water bottle D5032 02 Add 720 mL water bottle D5032 03 Add 720 mL water bottle NOTE The procedures below have been optimized for use with FRESH or FROZEN blood samples Anti coagulated blood or Buffy Coat can also be used However DNA yield will be reduced with time of storage In addition leukocytes or cultured cells may be used with this procedure DNA Yields From Various Starting Materials Species and Material Amount of Starting material Human Whole Blood Yield varies depending on the quantity of whit
25. in will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 50 mL centrifuge tube containing 12 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 5 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 12 mL of 70 ethanol and invert the tube 5 times to wash the DNA pellet Centrifuge at 2 000 x g for 5 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out the pellet Note If the resulting pellets are loose centrifuge at higher speed or with prolonged time Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 1mL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 28 of 45 M DNA Purification Protocol for Buffy Coat Prepared from 1 1 5 mL whole blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease f
26. ing 6 mL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 6 mL of 70 ethanol and invert the tube few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 600 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and overnight at room temperature to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 26 of 45 L DNA Purification Protocol for 12 mL Whole Blood Materials to be supplied by user Centrifuge capable of 2 000 x g Nuclease free 50 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 12 mL whole blood or bone marrow to a nuclease free 50 mL microcentrifuge tube containing 36 mL ERL Buffer Mix by inverting the tube 5 times Incubate 5 minutes at room temperature Invert the tube once every minute during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must
27. ion to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 20 of 45 I DNA Purification Protocol for 4 mL Whole Blood Materials to be supplied by user Centrifuge capable of 2 000 xg Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 4 mL whole blood or bone marrow to a nuclease free 15 mL microcentrifuge tube containing 12 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 300 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended Add 4 mL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells
28. l white pellet Pour out the supernatant and drain the tube briefly on an absorbent paper towel Add 800 uL of 70 ethanol and invert the tube a few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful not to pour out the pellet Invert the tube on an absorbent paper towel and air dry the pellet for 10 15 minutes Add 100 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour to rehydrate DNA Gently shake tube several times during incubation to disperse the DNA Some samples may need to incubate at room temperature overnight to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C Page 14 of 45 F DNA Purification Protocol for 1 mL Whole Blood Materials to be supplied by user Centrifuge capable of 14 000 x g Nuclease free 1 5 mL microcentrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 1 mL whole blood or bone marrow to a nuclease free 15 mL centrifuge tube containing 3 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at
29. le at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 34 of 45 Q DNA Purification Protocol for Cultured Cells 3 5 10 Materials to be supplied by user 10 11 12 13 Centrifuge capable of 2 000 xg Nuclease free 15 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C lsopropanol 70 ethanol This protocol is designed for isolating genomic DNA from 3 5 10 cultured cells Harvest the cells and transfer them with salt balanced buffer such as PBS to a15 mL microcentrifuge tube For adherent cells trypsinize the cells before harvesting Centrifuge at 500 x g for 3 minutes to pellet the cells Remove the cells and leave behind about 2500 uL residue liquid Vortex the cells to resuspend the cells in the residue liquid Make no cell clumps visible at this point Add 6000 uL of WTL Buffer to the re suspended cells and mix by pipetting The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps are cannot be seen Optional Add 30 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes Cool the sample to room temperature Add 2000uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30
30. liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended 10 11 12 13 14 15 16 17 18 Add 100uL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 0 5uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes Cool the sample to room temperature Add 33yuL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 1 5 mL centrifuge tube containing 100 uL of 100 isopr
31. nded white blood cell pellet before adding WTL buffer Vortex vigorously to completely resuspend white blood cell 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 The sample was not cooled to room temperature before adding PCP buffer Cool the sample to room temperature or chill on ice for at least 5 minutes before adding PCP buffer Low Ayeo Aogo Poor cell lysis due to incomplete mixing with Buffer WTL Repeat the procedure this time making sere to vortex the sample with Buffer WTL immediately and completely Hemoglobin remains Repeat the procedure this time making sure enough volume of ERL is used and white blood cell pellet is white in color PCP Buffer was not mixed Make sure that PCP buffer and cell with WTL buffer throughly lysate is mixed throughly DNA pellet was lost during isopropanol precipitation Be very careful not to lose the DNA when removing isopropanol or ethanol during precipitation and wash steps DNA Pelletis DNA pellet was over dried Rehydrate the DNA by incubating the difficult to DNA pellet with EB Buffer at 65 C dissolve for 1 hour and then leave the sample at room temperature or 4 C for overnight DNA pellet was not mixed Shake a few times during the well during rehydration step rehydration step Pa
32. od include any liquid residual into a 50 mL centrifuge tube Discard the supernatant and invert the tube onto a clean absorbent paper for 5 minutes make sure the DNA pellet remains inside the tube Air dry the DNA pellet until all liquid are evaporated Do not over dry the pellet because it is very difficult to dissolve the over dried DNA pellet Add 1 mL EB Buffer or TE Buffer vortex 5 seconds at lower speed Dissolve the DNA by incubating 1 hour at 65C and overnight at room temperature Page 42 of 45 Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water Tris HCI buffer or Elution Buffer as blank Dilute the DNA in TE buffer and calculate concentration 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 Aj6o A of 1 7 1 9 corresponds to 85 95 purity Expected yields range from 4 pg to 12 ug DNA per 250 uL whole blood depending on source of sample its age and the method of storage Yields are generally 5 fold higher with Buffy Coat samples Troubleshooting Guide Problem Probiem Possible Cause suggestions _ Low DNA Blood Sample contains too Draw new blood samples he F white blood cells Blood Blood sample is too oid is too old Try to use fresh blood if possible f to use fresh blood if possible Incompletely re suspe
33. of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended Add 6 mL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 30 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 2 0 mL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The 10 11 12 13 14 15 16 17 18 precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube contain
34. opanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 100uL of 70 ethanol and invert the tube 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 absorbent paper towel and air dry the pellet for 10 15 minutes Add 35 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix 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 Page 6 of 45 B DNA Purification Protocol for 200uL whole blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 1 5 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C lsopropanol 70 ethanol Add 200uL whole blood or bone marrow to a nuclease free 1 5 mL microcentrifuge tube containing 600uL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or P
35. pend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely resuspended Add 800uL WTL Buffer to the tube containing the resuspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 4 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 270 uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing 27 28 29 30 31 32 33 34 35 36 Centrifuge at 2 000 x g or higher for 5 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible repeat Step 8 incubate the tube in ice for 5 minutes and then repeat Step 9 Transfer the supernatant to a new nuclease free 15 mL centrifuge tube containing 800 uL of 100 isopropanol Do not transfer the protein pellet Gently mix the solution by inverting the tube 40 50 times Centrifuge at 2 000 x g for 3 minutes at room temperature DNA will be visible as a smal
36. per towel Add 1 3 mL 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 500 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and room temperature for overnight to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 30 of 45 Number of Cells l Isopropanol 70 EtOH Buffer EB N DNA Purification Protocol for Cultured Cells 0 5 1 x 10 Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 2 0 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C Isopropanol 70 ethanol 1 This protocol is designed for isolating genomic DNA from 0 5 1 million cultured cells 2 Harvest the cells and transfer them with salt balanced buffer such as PBS to a 2 0 mL microcentrifuge tube For adherent cells trypsinize the cells before harvesting 3 Centrifuge at 14 000 x g for 10 seconds to pellet the cells Remove the cells and leave behind about 25 uL residue liquid 4 Vortex the cells to resuspend the cells in the residue liquid Make no
37. precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat this step 11 Transfer the supernatant to a new 50 mL centrifuge tube containing12 mL of 100 isopropanol Add 20uL of glycogen 20mg mL per sample 12 Gently mix the solution by inverting the tube 30 40 times Centrifuge at 2000 x g for 5 minute at room temperature DNA will be visible as a small white pellet 13 Pour of the supernatant and drain the tube briefly on a clean absorbent paper towel Add 12mL of 70 ethanol and invert the tube a 5 times to wash the DNA pellet 14 Centrifuge at 2000 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 15 Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10 15 minutes 16 Add 400 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix 17 Incubate sample at 65 C for 1 hour and room temperature for overnight to Page 40 of 45 Q DNA Purification from large volume gt 1mL Clotted Blood 17 18 2 Homogenize the sample with a rotor stator homogenizer until the sample is uniformly homogenous 19 3 Add 3 volume of 1 x ERL buffer and mix by invert the tube 5 7 times 4 Incubate at RT for 5 minutes 5 Centrifuge at 2000 x g for 5 minutes 6 Discard the supernatant and leave the tube inverte
38. r 3 mL Whole Blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels lsopropanol 70 ethanol Add 3 mL whole blood or bone marrow to a nuclease free 15 mL centrifuge tube containing 9 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 150 200 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Vortex the tube vigorously until the cell pellet is completely resuspended Add 3 mL WTL Buffer to the tube containing the resuspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 15 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at
39. re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 3 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes Cool the sample to room temperature 10 11 12 13 14 15 16 17 18 19 20 Add 200uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 11 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 600uL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour of 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
40. ree 2 0 mL microcentrifuge tubes Water Bath preset at 37 C and 65 C Isopropanol 70 ethanol 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 1 Add 75 120 ul buffy coat preparation prepared from 1 5 mL whole blood to a nuclease free 2 mL microcentrifuge tube containing 400uL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation 2 Centrifuge at 14 000 x g for 30 seconds at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 15uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volume
41. riefly on an absorbent paper towel Add 5 mL of 70 ethanol and invert the tube few times to wash the DNA pellet Centrifuge at 2 000 x g for 2 minutes at room temperature Carefully pour out the ethanol Pellet may be very loose at this point so pour slowly and be careful Invert the tube on an adsorbent paper towel and air dry the pellet for 10 15 minutes Add 500 uL of Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 1 hour and overnight at room temperature to rehydrate DNA Gently shake tube several times during incubation to disperse DNA Store DNA at 2 8 C For long term storage store at 20 C Page 24 of 45 K DNA Purification Protocol for 6 mL Whole Blood Materials to be supplied by user Centrifuge capable of 2 000 xg Nuclease free 15 mL centrifuge tubes Water baths preset at 37 C and 65 C Paper towels Isopropanol 100 70 ethanol Add 6 mL whole blood or bone marrow to a nuclease free 50 mL microcentrifuge tube containing 18 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or instruction on Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 400 uL
42. rtexing Incubate in ice for 5 minutes 10 11 12 13 14 15 16 17 18 Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 1 5 mL centrifuge tube containing 300uL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel Add 300uL of 70 ethanol and invert the tube 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 uL of DNA rehydration solution Buffer EB and vortex for 1 minute to mix Incubate sample at 65 C for 10 min Some samples may need to incubate at 65 C for 1 2 hour to rehydrate DNA Store DNA at 2 8 C For long term storage store at 20 C Page 10 of 45 D DNA Purification Protocol for 600uL whole blood Materials to be supplied by user Microcentrifuge capable of 14 000 x g Nuclease free 2 0 mL microcentrifug
43. s ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 3 Vortex the tube vigorously until the white blood cells are completely resuspended 4 Add 1 3 mL WTL Buffer to the tube containing the resuspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen 5 Optional Add 5 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes 6 Cool the sample to room temperature 7 Add 433uL PCP Buffer to the cell lysate 10 11 12 13 14 15 16 17 18 Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vortexing Incubate in ice for 5 minutes Centrifuge at max speed for 3 minutes at room temperature The precipitated protein will form a tight dark brown pellet If the pellet is not tight or visible incubate the tube in ice for 5 minutes and repeat Step 9 Transfer the supernatant to a new nuclease free 2 0 mL centrifuge tube containing 1 3 mL of 100 isopropanol Gently mix the solution by inverting the tube 30 40 times Centrifuge at 14 000 x g for 1 minute at room temperature DNA will be visible as a small white pellet Pour out the supernatant and drain the tube briefly on a clean absorbent pa
44. uge tube containing 6 mL ERL Buffer Mix by inverting the tube a few times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 2 000 x g for 5 minutes at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 150 uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely resuspended Add 2 mL WTL Buffer to the tube containing the resuspended cells Pipet up and down to lyse the cells The solution should become very viscous If cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 10 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for approximately 10 minutes Cool the sample to room temperature Add 670 uL PCP Buffer to the cell lysate Vortex vigorously at high spe
45. w times Incubate 5 minutes at room temperature Invert the tube several times during incubation NOTE ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH O according to bottle label or Page 4 above before use Centrifuge at 14 000 x g for 30 seconds at room temperature Remove and discard as much supernatant as possible without disturbing the visible white pellet Leave about 15uL of residue liquid in the tube If the blood sample has been frozen repeat Steps 1 2 until the pellet is white Note If some red blood cells or cell debris are still visible along with the white blood cell pellet resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer Incubate 2 min at room temperature Then pellet the white blood cells by repeating Step 2 Vortex the tube vigorously until the white blood cells are completely re suspended Add 300uL WTL Buffer to the tube containing the re suspended cells Pipet up and down to lyse the cells The solution should become very viscous If the cell clumps are visible after mixing incubate the solution at 37 C until the clumps cannot be seen Optional Add 1 5 uL RNase A solution to the cell lysate Mix the sample by inverting the tube 20 25 times Incubate the mixture at 37 C for 5 10 minutes Cool the sample to room temperature Add 100uL PCP Buffer to the cell lysate Vortex vigorously at high speed for 30 seconds to mix Some protein clumps may be visible after vo
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