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Blood DNA Purification Kit - 30 mL - Protocol

1. Average Yield from 1 mL of whole blood 7 x 10 white blood cells 2ang DNA size Up to 200 kbp Average purity OD260 280 gt 1 7 Time to Complete 10 Purifications 45 60 minutes DNA rehydration Yield will vary depending on the type of blood processed Advantages e Fast and simple processing e DNA can be isolated and detected from as little as 300 uL of blood e Isolate high quality and high molecular weight genomic DNA e Recovered genomic DNA is compatible with various downstream applications Kit Components Component Product 52400 RBC Lysis Solution 100 mL Cell Lysis Solution 40 mL Proteinase K 2 5 mL DNA Rehydration Solution 12 mL Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers The kit contains a ready touse Proteinase K solution which is dissolved in a specially prepared storage buffer The Proteinase K is stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of Proteinase K storage at 2 8 C is recommended Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please con
2. decreased respectively Incubate at 65 C for 60 minutes to completely rehydrate DNA Ensure that the DNA is completely rehydrated Gentle pipetting as well as overnight incubation at room temperature may be used Note The prepared high yield DNA might be viscous due to the high concentration and molecular weight which can affect DNA analysis It is recommended to use a larger DNA Rehydration Solution volume and or longer rehydration time with gentle pipetting to ensure that no aggregates are formed Purification Procedure for 10 mL Blood Sample Add 30 mL of RBC Lysis Solution to a 50 mL centrifuge tube Add 10 mL of whole blood and mix by inverting 10 times Incubate at room temperature for 5 minutes and invert at least once during the incubation Centrifuge for 5 minutes at 2 000 x g to pellet white blood cells Carefully discard the supernatant by pipetting or pouring leaving about 200 uL of residual liquid Make sure not to disturb the white blood cells pellet Note For DNA isolation from blood containing Gram positive bacterial pathogens add 50 uL of lysozyme not provided and mix well by pipetting Incubate at 37 C for 1 hour NOTE incubation times may fluctuate between 0 5 and 2 hours depending on the bacterial strain being lysed After incubation proceed to step 6 Add 10 mL of Cell Lysis Solution Add 750 uL of Proteinase K vortex Proteinase K before use Pipette gently to resuspend the pellet into the added solu
3. resuspending in the DNA 65 C can be used if the sample is not completely Rehydration Solution rehydrated Protein carry over in the Make sure not to exceed the recommended input purified DNA amount of cells Low input Count cells to ensure that your sample has a high P enough amount of starting cells Exceeding the recommended input will increase the incoriiplete Iysis viscosity and cells will clump due to incomplete p y lysis resulting in a lower yield To avoid this cell Low yield counting of the blood sample is recommended Incomplete rehydration Do not over dry the DNA pellet before adding the DNA Rehydration Solution Mix well to resuspend the DNA before incubating at 65 C for the specified time Up to 1 hour incubation at 65 C may be used to ensure complete rehydration High A260 A280 RNA contamination RNase treatment of purified sample can be carried out to remove residual RNA DNA size is less than 50 kbp Blood samples should be stored at 4 C for few days or at 20 C if storage exceeds 5 days PN dagracanon Storage at 80 C is recommended for archival sample storage Vigorous handling of DNA during the different steps of the protocol can shear the DNA Avoid vigorous DNA shearing vortexing and use gentle pipetting in all of the mixing and resuspension steps Related Products Product Blood Genomic DNA Isolation Micro Kit 52100 Blood Genomic DNA Isolation Min
4. use e Blood Sample Fresh or frozen whole blood samples treated with the appropriate anticoagulant EDTA citrate or heparin may be used Better yield is obtained from fresh blood and EDTA is preferred for high molecular weight genomic DNA Blood can be collected on the desired anticoagulant and stored at 80 before genomic DNA purification e Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again e Thaw frozen blood samples quickly at 37 C with gentle agitation and keep on ice before starting the purification procedures e Vortex Proteinase K before use e Prepare the required volume of 70 ethanol e Preheat water bath or incubator to 56 C e Preheat 65 C incubator for the final rehydration step e For blood containing Gram positive bacterial pathogens prepare a 400 mg mL stock solution approximately 1 7 x 10 units mL of lysozyme as per supplier s instructions e Vigorous handling of DNA during the different steps of the protocol can shear the DNA Avoid vigorous vortexing and use gentle pipetting in all of the mixing and resuspension steps if higher molecular weight DNA is required A Purification Procedure for 300 pL Blood Sample r 2 3 4 5 Add 900 uL of RBC Lysis Solution to a 1 5 mL microcentrifuge tube Add 300 uL of whole blood and mix by inverting 10 times Incubate at room temp
5. 3430 Schmon Parkway gt N lt Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com Blood DNA Purification Kit Plus 30mL Product Insert Product 52400 Norgen s Blood DNA Purification Kit Plus 30 mL provides a fast and simple procedure for purifying high molecular weight genomic DNA from up to 10 mL of blood The kit allows for the isolation of genomic DNA from the blood of various species including humans Typical yields of genomic DNA will vary depending on the cell density of the blood sample Preparation time for a single sample is about 60 minutes Blood genomic DNA purified using Norgen s kit is of the highest quality and is compatible with a number of downstream applications including PCR Southern Blot analysis sequencing and microarray analysis Purification is based on the lysis of red blood cells RBC using the Norgen s RBC Lysis Solution followed by precipitating the white blood cells WBC Norgen s Cell Lysis Solution and Proteinase K are then added to the WBC pellet to lyse the cells and remove proteins Genomic DNA is then recovered by alcohol precipitation and resuspended in the DNA Rehydration Solution The size of the purified blood genomic DNA is up to 200 kpb with a typical Azgo Azgo ratio of gt 1 7 Specifications Kit Specifications Minimum blood input 0 3 mL Maximum blood input 10 mL
6. erature for 3 minutes and invert once during the incubation Centrifuge for 5 minutes at 2 000 x g to pellet white blood cells Discard the supernatant by pipetting leaving approximately 50 uL of residual liquid Make sure not to disturb the white blood cells pellet Note For DNA isolation from blood containing Gram positive bacterial pathogens add 5 uL of lysozyme not provided and mix well by pipetting Incubate at 37 C for 1 hour NOTE incubation times may fluctuate between 0 5 and 2 hours depending on the bacterial strain being lysed After incubation proceed to Step 6 oN 10 11 12 13 14 Centrifuge for 1 minute at 14 000 x g 16 17 18 is aPwons OOD 12 13 14 Add 300 uL of Cell Lysis Solution Add 22 5 uL of Proteinase K vortex Proteinase K before use Pipette gently to resuspend the pellet in the solutions Make sure that the pellet is completely dispersed Incubate at 56 C for 20 minutes Place the lysate tube in ice for 1 minute Add 375 uL of lsopropanol and invert 50 times to mix DNA threads or clump may be visible after mixing Centrifuge for 1 minute at 14 000 x g A white pellet of DNA may be visible Carefully discard the supernatant without disturbing the pellet Invert the tube on clean absorbent paper for 1 minute to drain residual isopropanol Add 900 uL of 70 ethanol and invert 10 times Carefully discard the supernatant Invert the tube upside down on clean ab
7. gal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Flow Chart Procedure for Purifying Blood Genomic DNA using Norgen s Blood DNA Purification Kit Plus 30 mL Desired volume of Blood in the appropriate tube size Add RBC Lysis Solution and mix let stand for 5 min SPIN Decant supernatant add Cell Lysis Solution and Proteinase K resuspend and incubate at 56 C for 20 30 min then 1 min on ice Add lsopropanol and invert to mix SPIN Add 70 Ethanol and invert to mix SPIN lt a N Dry add DNA Rehydration Solution and rehydrate Pure Blood Genomic DNA Volumes Required for Scaling Whole Blood Purification Protocols to Process Different Blood Input Volumes Blood volume pL 300 3 000 10 000 tTube size mL 1 5 15 50 RBC Lysis Solution uL 900 9 000 30 000 Cell Lysis Solution uL 300 3 000 10 000 Proteinase K uL 22 5 225 750 Isopropanol uL 375 3 500 11 000 70 ethanol uL 900 9 000 30 000 DNA Rehydration Solution uL 100 300 1 000 To isolate DNA from different volumes of blood than shown above use kit solution volumes that are proportional to the starting blood volume Use 50 mL centrifuge tube if the starting blood volume exceeds 3 5mL Lower volumes can be used to obtain higher DNA concentration however longer rehydration time may be required Notes prior to
8. i Kit 46300 Blood Genomic DNA Isolation Midi Kit 51400 Blood Genomic DNA Isolation Maxi Kit 31200 Dried Blood Spot Genomic DNA Isolation Kit 36000 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2013 Norgen Biotek Corp P152400 2
9. ified DNA sample may be stored at 4 C for a few days It is recommended that samples be placed at 20 C for long term storage or at 80 C for archival sample storage Troubleshooting Guide Problem Possible Cause Solution and Explanation The protocol is optimized for a cell density of 7 x 10 white blood cells per 1 mL of blood Exceeding To many cell were used the recommended input will increase the viscosity y and the cells will clump due to incomplete lysis Cells are not More lysis solution will be required To avoid this completely cell counting of the blood sample is recommended lysed i y a pear Pn A V Make sure to completely mix the lysate by gentle pse y pipetting No cell clumps should be visible after after the addition of the Aj mixing and the cell pellet should be completely Cell Lysis Solution and dislodged Proteinase K Samples were not Make sure to pipette well to resuspend DNA into resuspended after adding the DNA Rehydration Solution Avoid vigorous the DNA Rehydration vortexing if higher molecular weight DNA is Solution required Do not exceed the recommended air drying time Over drying will slow down the rehydration step and Samples are PNA peler overdrymg more incubation time at 65 C up to 1 hour may not completely b e required rehydrated F Samples were not Make sure to incubate the sample at 65 C for the incubated at 65 C after recommended time Up to 1 hour incubation at
10. l by pipetting Incubate at 37 C for 1 hour NOTE incubation times may fluctuate between 0 5 and 2 hours depending on the bacterial strain being lysed After incubation proceed to step 6 Add 3 mL of Cell Lysis Solution Add 225 uL of Proteinase K vortex Proteinase K before use Pipette gently to resuspend the pellet into the added solutions Make sure that the pellet is completely dispersed Incubate at 56 C for 25 minutes Place the lysate tube in ice for 2 minutes 11 Add 3 5 mL of lsopropanol and invert 50 times to mix DNA threads or clump may be visible after mixing Centrifuge for 3 minutes at 2 2 000 x g A white pellet of DNA may be visible Carefully discard the supernatant without disturbing the pellet Invert the tube on clean absorbent paper for 1 minute to drain residual isopropanol Add 9 mL of 70 ethanol and invert 10 times 15 16 17 18 O OP O oND 10 11 12 13 14 Centrifuge for 2 minutes at 2 2 000 x g 16 17 18 Centrifuge for 1 minute at 2 2 000 x g Carefully discard the supernatant Invert the tube upside down on clean absorbent paper for 10 minutes to drain residual ethanol and air dry Handle the tube carefully as the DNA pellet can dislodge Make sure not to over dry the DNA Add 300 uL of DNA Rehydration Solution and pipette gently to resuspend DNA Note For lower or higher DNA concentrations the volume of DNA Rehydration Solution can be increased or
11. sorbent paper for 5 minutes to drain residual ethanol and air dry Handle the tube carefully as the DNA pellet can dislodge Make sure not to over dry the DNA Add 100 uL of DNA Rehydration Solution and pipette gently to resuspend DNA Note For lower or higher DNA concentrations the volume of DNA Rehydration Solution can be increased or decreased respectively Incubate at 65 C for 5 minutes to completely rehydrate DNA Ensure that the DNA is completely rehydrated Gentle pipetting as well as overnight incubation at room temperature may be used Note The prepared high yield DNA might be viscous due to the high concentration and molecular weight which can affect DNA analysis It is recommended to use a larger DNA Rehydration Solution volume and or longer rehydration time with gentle pipetting to ensure that no aggregates are formed Purification Procedure for 3 mL Blood Sample Add 9 mL of RBC Lysis Solution to a 15 mL centrifuge tube Add 3 mL of whole blood and mix by inverting 10 times Incubate at room temperature for 5 minutes and invert at least once during the incubation Centrifuge for 5 minutes at 2 000 x g to pellet white blood cells Carefully discard the supernatant by pipetting or pouring leaving approximately 200 uL of residual liquid Make sure not to disturb the white blood cells pellet Note For DNA isolation from blood containing Gram positive bacterial pathogens add 20 uL of lysozyme not provided and mix wel
12. sult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Cell Lysis Solution contains guanidinium salts and should be handled with care Guanidinium salts forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with blood Customer Supplied Reagents and Equipment e Benchtop microcentrifuge variable speed up 14 000 x g or swing bucket centrifuge 2 2 000 x g Micropipettors and pipette tips 1 5 mL sterile microcentrifuge tubes 15 mL centrifuge tubes or 50 mL centrifuge tubes Isopropanol 70 ethanol does not contain other substances such as methanol or methylethylketone 56 C waterbath or incubator 65 C incubator Crushed ice 37 C incubator for blood containing Gram positive bacterial pathogens Lysozyme for blood containing Gram positive bacterial pathogens Procedure Please check your microcentrifuge or centrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rpm can be calculated using the formula RPM RCF 1 118 x 105 r where RCF required gravitational acceleration relative centrifu
13. tions Make sure that the pellet is completely dispersed Incubate at 56 C for 30 minutes Place the lysate tube in ice for 3 minutes Add 11 mL of Isopropanol and invert 50 times to mix DNA threads or clump may be visible after mixing Centrifuge for 5 minutes at 2 2 000 x g A white pellet of DNA may be visible Carefully discard the supernatant without disturbing the pellet Invert the tube on a clean absorbent paper for 1 minute to drain residual isopropanol Add 30 mL of 70 ethanol and invert 10 times Carefully discard the supernatant Invert the tube upside down on clean absorbent paper for 10 minutes to drain residual ethanol and air dry Handle the tube carefully as the DNA pellet can dislodge Make sure not to over dry the DNA Add 1 mL of DNA Rehydration Solution pipette gently to resuspend DNA Note For lower or higher DNA concentrations the volume of DNA Rehydration Solution can be increased or decreased respectively Incubate at 65 C for 60 minutes to completely rehydrate DNA Ensure that the DNA is completely rehydrated Gentle pipetting as well as overnight incubation at room temperature may be used Note The prepared high yield DNA might be viscous due to the high concentration and molecular weight which can affect DNA analysis It is recommended to use a larger DNA Rehydration Solution volume and or longer rehydration time with gentle pipetting to ensure that no aggregates are formed Storage of DNA The pur

Blood DNA Purification Kit - 30 mL - Protocol

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