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Table of Contents

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

(Bacteria, Plant, Animal, Blood)

Introduction Limitations of Use Features Applications Storage Quality Control

p2 p2 p3 p3 p3 p3

EZ-10 Spin Column Genomic DNA Minipreps Kit, Bacteria p4-9 EZ-10 Spin Column Genomic DNA Minipreps Kit, Plant p10-14
Version 4.7 Rev 02/16/2012

EZ-10 Spin Column Genomic DNA Minipreps Kit, Animal p15-23 EZ-10 Spin Column Genomic DNA Minipreps Kit, Blood p23-27

20 Konrad Crescent, Markham Ontario L3R 8T4 Canada Tel: (905) 474 4493, (800) 313 7224 Fax: (905) 474 5794 Email: order@biobasic.com Web: www.biobasic.com

EZ-10 Genomic DNA Kit Handbook

Introduction The EZ-10 Spin Column Kit system provides a fast, simple and efficient method for purification of genomic DNA from different sources such as Bacteria, Plant, Animal and Blood. DNA is selectively adsorbed in silica gel-based EZ-10 Spin Column while other components and impurities are washed away. The genomic DNA is eluted off the column and can be readily used in any downstream applications, including restriction enzyme digestion, PCR, Southern-blotting etc. The purification methods used in these protocols do not require use of phenol, chloroform, or CsCl. DNA is purified without an additional step of ethanol precipitation. Limitations of Use These kits are designed for research only. The purified DNA should not be used for live animal transfections. It is also not to be used for human diagnostic or drug production purposes.

Features Simple, fast and efficient Preparation of high quality genomic DNA from various sources High yield and reproducible No phenol chloroform extraction or ethanol precipitation required High capacity up to 10 g of DNA purified Applications Efficient of up to 10 g of genomic DNA purification from different sources. Storage With the exception of the Proteinase K, the kit may be stored at room temperature. Proteinase K should be stored at 4 C for short term or -20 C for long term. The kit is stable for 12 months at room temperature. For maximum stability, store all contents at 4 C. Quality Control Each lot of EZ-10 Spin Column kit is tested against predetermined specifications to ensure consistent product quality.

EZ-10 Genomic DNA Kit Handbook

EZ-10 Genomic DNA Kit Handbook

EZ-10 Spin Column Genomic DNA Minipreps Kit, Bacteria EZ-10 Spin Genomic DNA Minipreps Kit, Bacteria Digestion Solution (a) Wash Solution(b) Elution Buffer(c) Proteinase K(d) EZ-10 Column & 2.0-ml Collection Tube Protocol BS423 50 Preps 20 ml 12 ml 5 ml 2 mg 50 1 BS624 250 Preps 100 ml 2X30ml 25 ml 10 mg 250 1

proteinase K respectively. For long term storage store proteinase K solution at -20 C. Principle This kit is designed for fast isolation of genomic DNA from cells and bacteria. The kit contains a membrane embedded column for binding up to 10 g of genomic DNA. Nucleotides, proteins, salts, and other impurities are washed away. Purified genomic DNA can be used in most molecular biology experiments including restriction enzyme digestion, PCR, Southern-blotting etc. Procedures for Isolation of Genomic DNA from Cells and Bacteria 1. Sample Preparation A. Cell Cultures (1) Cells grown in suspension Spin appropriate number of cells (max. 5 million cells) at 2,500 x g (5,000 rpm) for 5 minutes at room temperature. Remove supernatant completely and discard. Wash the cell pellet twice with PBS and resuspend cells in 200 l cold TE, proceed to Step 2. (2) Cells grown in monolayer Aspirate the medium and wash cells with PBS. Remove PBS and apply trypsin solution to the cells. After cells have become detached, neutralize the trypsin with 2 volumes of medium.
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a) Digestion Solution may form a precipitate upon storage. If necessary, dissolve the precipitate by warming the solution at 37 C. b) Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS423, or 120 ml of 100% ethanol to 30 ml Wash Solution for BS624. For other volumes of Wash Solution, simply add enough ethanol to make a 4:1 ratio (volume of added ethanol: volume of Wash Solution = 4:1). c) Elution Buffer is 2.0mM Tris-HCl pH 8.08.5. Although Tris buffer pH 8.0 or water can be used, yield may be slightly lower. d) Before use, add 150 l, or 750 l of sterilized water to the tube containing 2 mg, or 10 mg of
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Centrifuge at 8,000 x g (10,000 rpm) for 5 minutes. Carefully remove supernatant and resuspend pellet in 200 l TE buffer, and continue immediately with Step 2. Notes: (a) If sample can not be used immediately for genomic DNA extraction, it is recommended to store at -20 C or -80 C for long-term. (b) Avoid repeated freezing and thawing of stored samples, since this leads to reduced DNA size and yield. B. Bacteria Collection Spin appropriate number of bacteria (about 106~107) at 6,000 x g (8,000 rpm) for 5 minutes at room temperature. Remove supernatant completely and discard, then resuspend cells in 200 l cold TE, proceed to Step 2. C. Paraffin Tissue (1) Excise 25~30 mg paraffin tissue with a clean, sharp scalpel. Transfer to a 1.5 ml Eppendorf tube. (2) Add 1.2 ml xylene (not included in the kit) to the tube, then vortex for 3 minutes. Xylene is used to remove paraffin. (3) Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature. (4) Remove the supernatant completely. Keep the
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(5)

(6)

(7) (8) (9)

pellet. Add 1.2 ml of 100% ethanol to the tube. Gently vortex for 1 minute. Incubate at room temperature for 1 minute. Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature. Discard supernatant completely. Repeat step 5 to 6. Incubate at 37 C for 10-15 minutes to remove residual ethanol. Resuspend the sample in 200 l TE buffer, and proceed to Step 2.

2. Add 400 l of Digestion Solution to 200 l sample from step 1. Mix well. Add 3 l Proteinase K solution (2mg/150l), and incubate at 55 C for 5 minutes. Notes: (a) Do not add proteinase K solution to Digestion Solution. (b) Incubation period depends on the nature of sample. For cell cultures, 5 minutes is generally enough to obtain complete lysate. For tissue samples it requires 3-5 hours. Longer period incubation even overnight incubation will not affect the result. 3. Add 260 l of 100% ethanol, and mix well. Apply the mixture onto an EZ-10 spin column

EZ-10 Genomic DNA Kit Handbook

that is placed in a 2.0 ml Collection Tube. Spin at 8,000 x g (10,000 rpm) for 2 minutes. 4. Discard the flow-through in the collection tube. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 2 minutes. 5. Repeat Step 4. 6. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 7. Place the EZ-10 column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50 C for 2 minutes. Incubate at 37 or 50 C could increase recovery yield. 8. Spin at 8,000 x g (10,000 rpm) for 2 minute to elute DNA from the column. 9. For long term storage, keep aliquots of purified genomic DNA at -20 C. 10. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. Troubleshooting Guide: EZ-10 Spin Column Genomic DNA Minipreps Kit, Bacteria 1. Low yield a. Improper storage of starting material Prepare fresh samples and use immediately.
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b. Too much or too LOW starting material Reduce or increase starting material accordingly. c. Incorrect preparation of buffers Each step has to be strictly followed. 2. RNA contamination Perform optional RNase treatment according to the protocol. 3. OD 260nm/280nm ratio outside 1.9-2.2 range If the ratio of OD260nm/280nm is greater than 2.2, there may be traces of ethanol present. If the ratio of OD260nm/280nm is smaller than 1.9, there may be chance of protein contamination. Make sure the sample is mixed well after proteinase K digestion. 4. DNA does not perform well a. DNA Shearing Avoid repeated freezing and thawing of starting material; if samples are too old, start with a new sample. b. Ethanol Carryover Spin additional minute before elution.

EZ-10 Genomic DNA Kit Handbook

EZ-10 Spin Column Genomic DNA Minipreps Kit, Plant EZ-10 Spin Genomic DNA Minipreps Kit, Plant RNase A (10 mg/ml)(a) PCL Solution PP Solution PB Solution Wash Solution(b) Elution Buffer(c) EZ-10 Spin Column & 2.0-ml Collection Tube Protocol BS425 50 Preps 150 l 15 ml 2 ml 20 ml 12 ml 5 ml 50 1 BS626 250 Preps 750 l 75 ml 10 ml 100 ml 2X30 ml 25 ml 250 1

enough ethanol to make a 4:1 ratio (volume of added ethanol: volume of Wash Solution = 4:1). c) Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can be used, yield may be 20% lower. Procedures for Isolation of Genomic DNA from Plants 1. Plant Tissue Sample Preparation Grind plant tissue under liquid nitrogen to a fine powder using a mortar and pestle. Transfer the powder and liquid nitrogen to 1.5 ml Eppendorf tube and allow liquid nitrogen to be evaporated. Do not allow the sample to thaw. Proceed immediately to Step 2. Note: (a) If sample can not be used immediately for genomic DNA extraction, it is recommended to store at -20 C for long-term use. (b) Avoid repeated freezing and thawing of stored samples, since this leads to reduced DNA size and yield. (c) Incubation period depends on the nature of sample. Longer period incubation even overnight incubation will not affect the result. (d) Better results could be achieved if starting material is less than 60 mg. In any case, the
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a) PCL Solution DOES NOT contain RNase A (100 g/ml). Please add entire contents of RNAse A into PCL Solution and store at 4 C for long term storage. PCL Solution may form a precipitate upon storage. If necessary, dissolve the precipitate by warming up to room temperature. b) Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS425, or 120 ml of 100% ethanol to 30 ml Wash Solution for BS626. For other volumes of wash solution, simply add

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2.

3.

4. 5.

6.

7. 8.

9.

quantity of samples should not exceed 100 mg for one EZ-10 Spin Column. Check the volume of grounded material, and add equal volume (approximately 150 l) of PCL Solution (Plant Cell Lysis Solution). Vortex and shake the tube several times. Incubate at 65 C for 20 minutes, vortex or pipette up and down to further remove any clumps. Clumped tissue will not lyse properly and will result in a lower yield of DNA. Add 25 l PP Solution. Mix well. Keep the solution on ice for 15 minutes. Centrifuge at 4 C, 8,000 x g (10,000 rpm) for 5 minutes. Apply the clear lysate to an EZ-10 Spin Column. Add 300 l PB buffer to the EZ-10 Spin Column. Mix gently by inverting the tube. Incubate the mixture for 3 minutes at room temperature. During incubation, mix occasionally by inverting the tube. Centrifuge at 4 C, 8,000 x g (10,000 rpm) for 2 minutes. Discard the flow-through in the Collection Tube. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 2 minutes. Repeat Step 8.

10. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 11. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate at RT for 2 or 3 minutes. Incubating the tube at 37 or 50 C for 2 minutes may increase recovery yield. 12. Spin at 8,000 x g (10,000 rpm) for 2 minutes to elute DNA from the column. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). 13. For long term storage, keep aliquots of purified genomic DNA at -20 C. 14. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. Troubleshooting Guide: EZ-10 Spin Column Genomic DNA Minipreps Kit, Plant 1. Low yield There are a number of variables that can cause low yield. a. Inefficient homogenization. Ensure that the material is completely disrupted. If
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necessary, increase time of homogenization. b. Low DNA content of plant tissue. Increase the amount of starting material to 200 mg. 2. RNA contamination RNase activity is weakened or lost. Add 30% additional RNAse A, and store solution at 4 C. 3. OD 260nm/280nm ratio outside 1.9-2.2 range If the ratio of OD260nm/280nm is greater than 2.2, there may be traces of ethanol present. If the ratio of OD260nm/280nm is smaller than 1.9, there is a chance of protein contamination. Make sure the sample is mixed well after PCL Solution. EZ-10 Spin Column Genomic DNA Minipreps Kit, Animal

EZ-10 Spin Genomic DNA Minipreps, Animal ACL Solution(a) PBS Solution AB Solution Proteinase K(b) Wash Solution(c) Elution Buffer(d) EZ-10 Spin Column & 2.0-ml Collection Tube Protocol

BS427 50 Preps 20 ml 75 ml 20 ml 20 mg 12 ml 5 ml 50 1

BS628 250 Preps 100 ml 2x200 ml 100 ml 100 mg 2X30 ml 50 ml 250 1

a) ACL Solution may form a precipitate upon storage. If necessary, dissolve the precipitate by warming the solution at 37 C. b) Before use, add 1 ml or 5 ml of sterilized water to the tube containing 20 mg or 100 mg of Proteinase K. Keep solution at -20 C for long term. c) Before use, add 48 ml of 100% ethanol to 12 ml Wash Solution for BS427 or 120 ml of 100% ethanol to 30 ml Wash Solution for BS628. For other volumes of wash solution, simply add enough ethanol to make a 4:1 ratio (volume of added ethanol: volume of Wash Solution = 4:1).

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d) Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 10% lower. Procedures for Isolation of Genomic DNA from Animal For Animal Tissue 1. Cut up to 30 mg of tissue and place in a 1.5 ml centrifuge tube. 2. Add 300 l of ACL Solution (Animal Cell Lysis Solution) to 1.5 ml centrifuge tubes and 20 l Proteinase K. 3. Incubate at 55 C until the tissue is completely lysed (usually 1-3 hours). Vortex occasionally. Incubate in shaking water bath can reduce lysis time. 4. Cool to room temperature. Vortex for 20 seconds and Centrifuge 10,000 x g (12,000 rpm) for 5 minutes. 5. Pipette 300 l of supernatant into an EZ-10 Spin Column (if pellet not visible, repeat previous step) and add 300 l of AB Solution. Mix by occasionally inverting tube, and keep for 2 minutes. 6. Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the flow-through.
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7. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 2 minutes. 8. Repeat Step 7. 9. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 10. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50 C for 2 minutes. Incubating at 37 or 50 C could increase recovery yield. 11. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column. 12. For long term storage, keep aliquots of purified genomic DNA at -20 C. 13. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. For Rodent Tail 1. Place numbered 1.5 ml centrifuge tubes on dry ice. 2. Cut 0.5 cm to 1 cm from ends of tails and place in tubes.
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3. Add 300 l of ACL Solution to 1.5 ml centrifuge tubes and 20 l of Proteinase K. 4. Incubate at 55 C overnight with rocking; or for several hours with occasional mild vortexing every 15 minutes. 5. Cool to room temperature. Vortex for 20 seconds and centrifuge at 10,000 x g (12,000 rpm) for 5 minutes. 6. Pipette 300 l of supernatant into an EZ-10 Spin Column (if pellet not visible, repeat previous step) and add 300 l of AB Solution. Mix by occasionally inverting tube, and keep for 2 minutes. 7. Centrifuge 2,000 x g (4,000 rpm) for 2 minutes and discard the flow-through. 8. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute. 9. Repeat Step 8 10. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 11. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50 C for 2 minutes. Incubating at 37 or 50 C could increase recovery yield.

12. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column. 13. For long term storage, keep aliquots of purified genomic DNA at -20 C. 14. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. For Cultured Animal Cell 1. Centrifuge the appropriate number of cells (>5x106) for 5 minutes at 200 x g (1,200 rpm). 2. Resuspend pellet in 500 l of PBS Solution. 3. Wash the cells 2 times with PBS Solution. 4. Resuspend pellet in 300 l of ACL solution buffer. 5. Add 20 l of Proteinase K. 6. Incubate at 55 C for 10 minutes. 7. Cool to room temperature. Vortex for 20 seconds and centrifuge 10,000 x g (12,000 rpm) for 5 minutes. 8. Pipette 200 l of supernatant into an EZ-10 Spin Column (if pellet not visible, repeat previous step) and add 200 l AB Solution. Mix by

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occasionally inverting tube, and keep for 2 minutes. 9. Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the flow-through. 10. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute. 11. Repeat Step 10. 12. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 13. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at RT for 2 minutes. To incubate at 37 or 50 C could increase recovery yield. 14. Spin at 8,000 x g (10,000 rpm) for 2 minutes to elute DNA from the column. 15. For long term storage, keep aliquots of purified genomic DNA at -20 C. 16. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb.

From Paraffin Tissue 1. Excise 25~30 mg paraffin tissue with a clean, sharp scalpel and transfer to a 1.5 ml Eppendorf tube. 2. Add 1.2 ml xylene (not included in the kit) to the tube, then vortex for 3 minutes. Xylene is used to remove paraffin. 3. Centrifuge at 10,000 x g (12,000 rpm) for 5 minute at room temperature. 4. Remove the supernatant completely. Keep the pellet. 5. Add 1.2 ml 100% of ethanol to the tube. Gently vortex for 1 minute. Incubate at room temperature for 1 minute. 6. Centrifuge at 10,000 x g (12,000 rpm) for 5 minute at room temperature. Discard supernatant completely. 7. Repeat step 4 to 6. 8. Incubate at 37 C for 10-15 minutes to remove residual ethanol. 9. Resuspend the sample in 200 l TE buffer, and proceed immediately to Step 10. 10. Add 300 l of ACL Solution (Animal Cell Lysis Solution) and 20 l Proteinase K. 11. Incubate at 55 C until the tissue is completely lysed (usually 1-3 hours). Vortex occasionally. Incubation in shaking water bath can reduce lysis time.

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12. Cool to room temperature. Vortex for 20 seconds and centrifuge at 10,000 x g (12,000 rpm) for 5 minutes. 13. Pipette 300 l of supernatant (if pellet not visible, repeat previous step) into a new tube and add 300 l of AB Solution. Mix by inverting the tube occasionally, and let the tube stand for 2 minutes. Apply entire contents onto an EZ-10 Spin Column. 14. Centrifuge at 2,000 x g (4,000 rpm) for 2 minutes and discard the flow-through. 15. Add 500 l of Wash Solution, and spin at 6,000 x g (8,000 rpm) for 1 minute. 16. Repeat Step 15. 17. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution. 18. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50 C for 2 minutes. Incubating at 37 or 50 C could increase recovery yield. 19. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column. 20. For long term storage, keep aliquots of purified genomic DNA at -20 C.

21. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. EZ-10 Spin Column Genomic DNA Minipreps Kit, Blood EZ-10 Spin Genomic DNA Minipreps Kit, Blood TBP Buffer TBM Buffer(a) TE (pH 8.0) Proteinase K(b) Wash Solution(b) Elution Buffer(c) EZ-10 Spin Column & 2.0-ml Collection Tube Protocol BS483 50 Preps 120 ml 25 ml 15 ml 2 mg 12 ml 5 ml 50 1 BS684 250 Preps 5X120 ml 125 ml 75 ml 10 mg 2X30ml 25 ml 250 1

(a) TBM Buffer may form a precipitate upon storage. If necessary, dissolve the precipitate by warming at 37 C. (b) Before use, add 150 l or 750 l of sterilized water to the tube containing 2 mg or 10 mg of Proteinase K respectively. Keep at -20 C for long term storage. Before use, add 48 ml of
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100% ethanol to 12 ml Wash Solution for BS483, or 120ml of 100% ethanol to 30ml Wash Solution for BS684. For other volumes of wash solution, simply add enough ethanol to make a 4:1 ratio (volume of added ethanol: volume of Wash Solution = 4:1). (c) Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 20% lower. Storage of Blood Whole blood samples treated with EDTA, ACD or heparin can be used, and may be either fresh or frozen. For short term storage (up to 10 days), collect blood in tubes containing EDTA as an anticoagulant, and store tubes at 2-8oC. It is recommended to store blood sample less than 3 days as DNA degradation may occur. For long term storage, collect blood in tubes containing a standard anticoagulant (preferably EDTA if high molecular weight DNA is required) and store -80oC. Blood Collection and Treatment For every 6ml of whole blood sample, add 1 ml of anticoagulant (0.5M EDTA pH 8.0, or ACD, 0.48% Citric Acid, 1.32% Sodium Citrate, 1.47% Glucose).

Procedures for Extraction Genomic DNA from Blood 1. Harvest 0.5 ml of whole blood in 2.0 ml centrifuge tube. Add 0.8 ml TBP Buffer to the tube. Vortex gently and let the tube stand for 1 min at room temperature. The erythrocytes should be lsyed and solution should appear clear red. 2. Spin at 2,000 x g (4,000 rpm) for 3 minutes. Discard supernatant. 3. Repeat Step 2. If the supernatant and blood pellet remain red in color, repeat step 2. If the blood pellet looks mauve or colorless, dissolve the pellet in 200 l TE buffer and continue with step 4. 4. Add 0.5 ml TBM Buffer to the centrifuge tube. Vortex the tube vigorously and then add 3 l Proteinase K. Incubate at 55 C for 30 minutes. 5. If insoluble material is visible, centrifuge for 2 minutes at 2,500 x g (5,000 rpm). Transfer supernatant to another 2.0 ml centrifuge tube and add 260 l absolute ethanol. 6. Apply the mixture to EZ-10 column that is in a 2.0 ml Collection Tube. Spin at 8,000 x g (10,000 rpm) for 2 minutes. Discard the flow-through in the collection tube. 7. Add 500 l of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 1 minute. 8. Repeat Step 7.

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9. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove any residual amount of Wash Solution. 10. Place the column into a clean 1.5 ml Eppendorf tube. Add 30-50 l Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50 C for 2 minutes. Incubating at 37 or 50 C could increase recovery yield. 11. Spin at 8,000 x g (10,000 rpm) for 1 minute to elute DNA from the column. 12. For long term storage, keep aliquots of purified genomic DNA at -20 C. 13. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 g). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb. Troubleshooting Guide 1. Low yield There are number of variables that can cause low yield a. Each step has to be strictly followed. b. Make sure column binding capacity 10 g is not exceeded. 2. RNA contamination

RNase activity is weakened or lost. Add 30% additional RNAse A, and store Solution at 4 C. 3. Sample floats upon loading in agarose gel The sample contains ethanol from washing step. Discard the liquid waste from the collection tube after washing step, and spin again for additional two minutes. Before elution step, incubate the column at 50 C for ~5 min and allow ethanol to evaporate completely.

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