EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

Transcription

1 EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03

2 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications Storage Quality Control EZ-0 Spin Column Genomic DNA Minipreps Kit, Bacteria Principle Procedures for Isolation of Genomic DNA from Cells or Bacteria EZ-0 Spin Column Genomic DNA Minipreps Kit, Plant Procedures for Isolation of Genomic DNA from Plants EZ-0 Spin Column Genomic DNA Minipreps Kit, Animal Procedures for Isolation of Genomic DNA from Animal For Animal Tissue For Rodent Tail For Cultured Animal Cell From Paraffin Tissue EZ-0 Spin Column Genomic DNA Minipreps Kit, Blood Storage of Blood Blood Collection and Treatment Procedures for Extraction Genomic DNA from Blood Introduction EZ-0 Spin Column Kits provide a fast, simple and efficient method for purification of genomic DNA from various sources such as Bacteria, Plant tissue, Animal tissue, Cells and Blood. By taking the advantage of silica-based DNA purification technology, DNA is selectively adsorbed in silica-based membrane embeded in EZ-0 Spin Column. Other components and impurities flow through the column or are washed away during wash steps. Genomic DNA is then eluted off the column and can be readily used in most downstream applications, including restriction enzyme digestion, PCR, Southern-blotting etc. The purification procedure using in these kits do not require use of hazardous compound such as phenol, chloroform, or CsCl. DNA is purified without additional steps of ethanol precipitation. Limitations of Use These kits are designed for research use only. 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 0 µg of DNA per column. Applications Obtain up to 0 µg of genomic DNA purification from various sources. Storage All components except Proteinase K could be stored at room temperature. Proteinase K should be kept at 4ºC for short term or -0ºC for long term storage. Kits are stable for months at room temperature after received. For maximum stability, store all contents at 4ºC. Quality Control Each lot of EZ-0 Spin Column kit is tested against predetermined specifications to ensure consistent product quality.

3 EZ-0 Genomic DNA Kit Handbook EZ-0 Spin Column Genomic DNA Minipreps Kit, Bacteria Component BS43, 50 Preps BS64, 50 Preps (a) Digestion Solution 0 ml 00 ml Wash Solution Elution Buffer Proteinase K EZ-0 Column (with 0-ml Collection Tube) Protocol (b) (c) (d) ml x 30 ml 5 ml 5 ml mg 50 0 mg Notes: a. Digestion Solution may form precipitates upon storage. Dissolve the precipitate by warming the solution at 37ºC if necessary. b. Before use, add 48 ml of 00% ethanol to ml Wash Solution for BS43, or 0 ml of 00% ethanol to 30 ml Wash Solution for BS6 For other volumes of Wash Solution, simply add ethanol to make a 4: ratio (volume of added ethanol:volume of Wash Solution = 4:). c. The recipe of Elution Buffer is 0 mm Tris-HCl, ph 0 Water can also be used but yield may be slightly lower. d. Before use, add 50 µl, or 750 µl of sterilized water to the tube containing mg, or 0 mg of proteinase K, respectively. For long term storage, proteinase K solution should be kept at -0ºC. Principle This kit is designed for rapid isolation of genomic DNA from cells and bacteria. The kit contains a membrane embedded column for binding up to 0 µg of genomic DNA. 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 or Bacteria. Sample Preparation. A. Cell Cultures () Cells grown in suspension 50 Spin appropriate number of cells (not exceed 5 x 06 cells) at,500 x g (5,000 rpm) for 5 minutes at room temperature. Remove supernatant completely. Wash the cell pellet twice with PBS Buffer (not provided with kit) and resuspend cells in 00 µl cold TE Buffer (not provided with kit), proceed to Step () Cells grown in monolayer Aspirate the medium and wash cells with PBS Buffer. Remove PBS and apply trypsin solution to the cells. After cells have become detached, neutralize the trypsin with volumes of medium. Centrifuge at 8,000 x g (0,000 rpm) for 5 minutes. Carefully remove supernatant and resuspend pellet in 00 µl TE buffer, and continue with Step If following steps could not be performed immediately, it is safe to pause here and it is recommended to store the lysate at -0 ºC or -80 ºC. 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 06~07) at 6,000 x g (8,000 rpm) for 5 minutes at room temperature. Remove supernatant completely and resuspend cells in 00 µl cold TE Buffer (not provided with kit), proceed to Step C. Paraffin Tissue () Excise 5~30 mg paraffin tissue with a clean, sharp scalpel. Transfer to a.5 ml Eppendorf tube. () Add. ml xylene (not provided with kit, Xylene is used to remove paraffin) to the tube, vortex for 3 minutes. (3) Centrifuge at 0,000 x g (,000 rpm) for 5 minutes at room temperature. (4) Discard the supernatant and keep the pellet. (5) Add. ml of 00% ethanol to the tube. Gently vortex for minute. Incubate at room temperature for minute. (6) Centrifuge at 0,000 x g (,000 rpm) for 5 minutes at room temperature. Discard supernatant. (7) Repeat step 5 to (8) Incubate at 37 ºC for 0-5 minutes to remove residual ethanol. 3 4

4 EZ-0 Genomic DNA Kit Handbook (9) Resuspend the sample in 00 µl TE buffer, and proceed to Step Add 400 µl of Digestion Solution to 00 µl sample from step. Mix well. Add 3 µl of Proteinase K solution ( mg/50 µl) to sample and incubate at 55 ºC for 5 minutes. Do not add proteinase K solution directly to Digestion Solution. 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 -5 hours. Longer period incubation even overnight will not affect the result. If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step Add 60 µl of 00% ethanol, and mix well. Apply the mixture onto an EZ-0 spin column that is placed in a 0 ml Collection Tube. Spin at 8,000 x g (0,000 rpm) for minutes. Discard the flow-through in the collection tube. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minutes. Repeat Step Discard flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the EZ-0 column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minute to elute DNA from the column.. Low yield a. Improper storage of starting material >>>Prepare fresh samples and use immediately. b. Too much or too less starting material >>>Reduce or increase starting material accordingly. c. Incorrect preparation of buffers >>>Each step has to be strictly followed. RNA contamination Perform optional RNase treatment according to the protocol. OD60nm/OD80nm ratio outside.7-.9 range If the ratio of OD60nm/OD80nm is greater than.9, there may be traces of RNA contamination. If the ratio of OD60nm/OD80nm is smaller than.7, there may be protein contamination. Make sure the sample is mixed well after proteinase K digestion. 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 steps before elution For long term storage, keep aliquots of purified genomic DNA at -0 ºC. 0. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel. 5 6

5 EZ-0 Genomic DNA Kit Handbook EZ-0 Spin Column Genomic DNA Minipreps Kit, Plant Component BS45, 50 Preps BS66, 50 Preps (a) RNase A (0 mg/ml) 50 µl 750 µl PCL Solution PP Solution PB Solution Wash Solution Elution Buffer EZ-0 Column (with 0-ml Collection Tube) Protocol (b) (c) 5 ml 75 ml ml 0 ml 0 m ml 5 ml ml x 30 ml 5 ml 50 Notes: a. PCL Solution DOES NOT contain RNase A (00 µg/ml). Please add entire contents of RNAse A into PCL Solution and store at 4ºC. PCL Solution may form precipitates upon storage. If necessary, dissolve the precipitate by warming up to room temperature. mg, or 0 mg of proteinase K, respectively. For long term storage, proteinase K solution should be kept at -0ºC. b. Before use, add 48 ml of 00% ethanol to ml Wash Solution for BS45, or 0 ml of 00% ethanol to 30 ml Wash Solution for BS6 For other volumes of wash solution, simply add enough ethanol to make a 4: ratio (volume of added ethanol:volume of Wash Solution = 4:). c. Elution Buffer is 0 mm Tris-HCl ph 0~ Although TE buffer ph 0 or water can be used, yield may be 0% lower. Procedures for Isolation of Genomic DNA from Plants. Plant Tissue Sample Preparation: Grind plant tissue under liquid nitrogen to a fine powder using a mortar and pestle. Transfer the powder immediately after liquid nitrogen to be evaporated to.5 ml Eppendorf tube. Do not allow the sample to thaw. Proceed immediately to Step If following steps could not be performed immediately, it is recommended to pause here and store the powder at -0 ºC. Avoid repeated freezing and thawing of stored samples, since this leads to reduced DNA size and yield. Incubation period depends on the nature of sample. Longer period incubation even overnight will not affect the result. Better results could be achieved if starting material is less than 60 mg. The amount of samples should not exceed 00 mg per EZ-0 Spin Column. Check the volume of grounded material, and add equal volume (approximately 50 µl) of PCL Solution (Plant Cell Lysis Solution). Vortex and shake the tube several times. Incubate at 65ºC for 0 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. If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step Add 5 µl PP Solution. Mix well. Keep the solution on ice for 5 minutes. Centrifuge at 4 ºC, 8,000 x g (0,000 rpm) for 5 minutes. Apply the clear lysate to an EZ-0 Spin Column. Add 300 µl PB buffer to the EZ-0 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 ºC, 8,000 x g (0,000 rpm) for minutes. 7 8

6 EZ-0 Genomic DNA Kit Handbook 0.. Discard the flow-through in the Collection Tube. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minutes. Repeat Step Discard flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minutes to elute DNA from the column. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). For long term storage, keep aliquots of purified genomic DNA at -0ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.. Low yield There are a number of variables that can cause low yield. a. Inefficient homogenization. >>>Ensure that the material is completely disrupted. If necessary, increase time of homogenization. b. Low DNA content of plant tissue. >>>Increase the amount of starting material to 00 mg. RNA contamination RNase activity is weakened or lost. Add 30% additional RNAse A, and store solution at 4 ºC. OD60nm/OD80nm ratio outside.7-.9 range EZ-0 Spin Column Genomic DNA Minipreps Kit, Animal Component BS47, 50 Preps BS68, 50 Preps (a) ACL Solution 0 ml 00 ml PBS Solution AB Solution Proteinase K Wash Solution Elution Buffer EZ-0 Column (with 0-ml Collection Tube) Protocol (b) (c) (d) 75 ml x 00 ml 0 ml 00 ml 0 mg ml 5 ml mg x 30 ml 5 ml 50 Notes: a. ACL Solution may form precipitates upon storage. If necessary, dissolve the precipitate by warming the solution to 37ºC. b. Before use, add ml or 5 ml of sterilized water to the tube containing 0 mg or 00 mg of Proteinase K. Keep solution at -0ºC. c. Before use, add 48 ml of 00% ethanol to ml Wash Solution for BS47 or 0 ml of 00% ethanol to 30 ml Wash Solution for BS6 For other volumes of wash solution, simply add enough ethanol to make a 4: ratio (volume of added ethanol:volume of Wash Solution = 4:). d. Elution Buffer is 0 mm Tris-HCl ph 0~ Although TE buffer ph 0 or water can be used, yield is generally 0% lower. If the ratio of OD60nm/OD80nm is greater than.9, there may be traces of RNA contamination. If the ratio of OD60nm/OD80nm is smaller than.7, there is a chance of protein contamination. Make sure the sample is mixed well after PCL Solution. 9 0

7 EZ-0 Genomic DNA Kit Handbook Procedures for Isolation of Genomic DNA from Animal For Animal Tissue. Cut up to 30 mg of tissue and place in a.5 ml centrifuge tube. Add 300 µl of ACL Solution (Animal Cell Lysis Solution) to.5 ml centrifuge tubes and 0 µl of Proteinase K. Incubate at 55ºC until tissues are completely lysed (usually -3 hours). Vortex occasionally. Incubate in shaking water bath can reduce lysis time.. For Rodent Tail Place numbered.5 ml centrifuge tubes on dry ice. Cut 0.5 cm to cm from ends of tails and place in tubes. Add 300 µl of ACL Solution to.5 ml centrifuge tubes and then add 0 µl of Proteinase K. Incubate at 55 ºC overnight with rocking; or for several hours with occasional mild vortexing every 5 minutes If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step Cool to room temperature. Vortex for 0 seconds and centrifuge 0,000 x g (,000 rpm) for 5 minutes. Pipette 300 µl of supernatant to a new Eppendorf tube, add 300 µl of AB Solution. Mix by occasionally inverting tube, and keep for minutes. Then load all the solution to a EZ-0 Spin Column. Centrifuge at,000 x g (4,000 rpm) for minutes and discard the flow-through. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minutes. Repeat Step Discard flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minute to elute DNA from the column. For long term storage, keep aliquots of purified genomic DNA at -0 ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarose gel If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step Cool to room temperature. Vortex for 0 seconds and centrifuge at 0,000 x g (,000 rpm) for 5 minutes. Pipette 300 µl of supernatant into to a new Eppendorf tube, add 300 µl of AB Solution. Mix by occasionally inverting tube, and keep for minutes. Then load all the solution to a EZ-0 Spin Column. Centrifuge,000 x g (4,000 rpm) for minutes and discard the flow-through. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minute. Repeat Step 8 Discard flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minute to elute DNA from the column. For long term storage, keep aliquots of purified genomic DNA at -0 ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel.

8 EZ-0 Genomic DNA Kit Handbook For Cultured Animal Cell Centrifuge the appropriate number of cells (>5x06) for 5 minutes at 00 x g (,00 rpm). Resuspend pellet in 500 µl of PBS Solution. Wash the cells times with PBS Solution. Resuspend pellet in 300 µl of ACL solution buffer. Add 0 µl of Proteinase K, mix well and Incubate at 55 ºC for 0 minutes. If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step Cool to room temperature. Vortex for 0 seconds and centrifuge 0,000 x g (,000 rpm) for 5 minutes. Pipette 00 µl of supernatant to a new Eppendorf tube, add 00 µl of AB Solution. Mix by occasionally inverting tube, and keep for minutes. Then load all the solution to a EZ-0 Spin Column. Centrifuge at,000 x g (4,000 rpm) for minutes and discard the flow-through. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minute. Repeat Step Discard flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minutes to elute DNA from the column. For long term storage, keep aliquots of purified genomic DNA at -0 ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel From Paraffin Tissue Excise 5~30 mg paraffin tissue with a clean, sharp scalpel and transfer to a.5 ml Eppendorf tube. Add. ml xylene (not included in the kit) to the tube, then vortex for 3 minutes. Xylene is used to remove paraffin. Centrifuge at 0,000 x g (,000 rpm) for 5 minute at room temperature. Discard the supernatant and keep the pellet. Add. ml 00% of ethanol to the tube. Gently vortex for minute. Incubate at room temperature for minute. Centrifuge at 0,000 x g (,000 rpm) for 5 minute at room temperature. Discard supernatant. Repeat step 4 to Incubate at 37 ºC for 0-5 minutes to remove residual ethanol. Resuspend the sample in 00 µl TE buffer, and proceed immediately to Step 0. Add 300 µl of ACL Solution (Animal Cell Lysis Solution) and then add 0 µl of Proteinase K. If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step. Incubate at 55 ºC until the tissue is completely lysed (usually -3 hours). Vortex occasionally. Incubation in shaking water bath can reduce lysis time. Cool to room temperature. Vortex for 0 seconds and centrifuge at 0,000 x g (,000 rpm) for 5 minutes. Pipette 300 µl of supernatant to a new Eppendorf tube, add 300 µl of AB Solution. Mix by occasionally inverting tube, and keep for minutes. Then load all the solution to a EZ-0 Spin Column. Centrifuge at,000 x g (4,000 rpm) for minutes and discard the flow-through. Add 500 µl of Wash Solution, and spin at 6,000 x g (8,000 rpm) for minute. 3 4

9 EZ-0 Genomic DNA Kit Handbook Repeat Step Discard the flow-through and spin at 8,000 x g (0,000 rpm) for an additional minute to remove residual amount of Wash Solution. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase Spin at 8,000 x g (0,000 rpm) for minute to elute DNA from the column 0. For long term storage, keep aliquots of purified genomic DNA at -0 ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel. EZ-0 Spin Column Genomic DNA Minipreps Kit, Blood Component BS483, 50 Preps BS Preps TBP Buffer 0 ml 5 x 0 ml TBM Buffer TE (ph 0) Proteinase K Wash Solution Elution Buffer (a) (b) (c) (d) 5 ml 5 ml 5 ml 75 ml mg ml 5 ml 0 mg x 30 ml 5 ml EZ-0 Column (with 0-ml Collection Tube) Protocol Notes: a. TBM Buffer may form a precipitate upon storage. If necessary, dissolve the precipitate by warming at 37 ºC. b. Before use, add 50 µl or 750 µl of sterilized water to the tube containing mg or 0 mg of Proteinase K respectively. Keep at -0 ºC for long term storage. c. Before use, add 48 ml of 00% ethanol to ml Wash Solution for BS483, or 0ml of 00% ethanol to 30ml Wash Solution for BS68 For other volumes of wash solution, simply add enough ethanol to make a 4: ratio (volume of added ethanol:volume of Wash Solution = 4:). d. Elution Buffer is 0 mm Tris-HCl ph 0~ Although TE buffer ph 0 or water can be used, yield is generally 0% 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 0 days), collect blood in tubes containing EDTA as an anticoagulant, and store tubes at -8 ºC. 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 at -80 ºC. 5 6

10 EZ-0 Genomic DNA Kit Handbook Blood Collection and Treatment For every 6 ml of whole blood sample, add ml of anticoagulant (0.5M EDTA ph 0, or ACD, 0.48% Citric Acid,.3% Sodium Citrate,.47% Glucose). Procedures for Extraction Genomic DNA from Blood. Harvest 0.5 ml of whole blood in 0 ml centrifuge tube. Add 0.8 ml TBP Buffer to the tube. Vortex gently and let the tube stand for min at room temperature. The erythrocytes should be lysed and solution should appear clear red. Spin at,000 x g (4,000 rpm) for 3 minutes. Discard supernatant. Repeat Step If the supernatant and blood pellet remain red in color, repeat step If the blood pellet looks mauve or colorless, dissolve the pellet in 00 µl TE buffer and continue with step Add 0.5 ml TBM Buffer to the centrifuge tube. Vortex the tube vigorously and then add 3 µl Proteinase K. Incubate at 55 ºC for 30 minutes. If RNA-free genomic DNA is required, add 0 µl RNase A (0 mg/ml, not temperature before continuing with step. For long term storage, keep aliquots of purified genomic DNA at -0 ºC. Measure DNA quantity by UV absorption at A60 (.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel. Low yield There are a number of variables that can cause low yield. a. Each step has to be strictly followed. b. Make sure column binding capacity 0 µg is not exceeded. RNA contamination RNase activity is weakened or lost. Add 30% additional RNAse A, and store solution at 4 ºC. 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 ºC for ~5 min and allow ethanol to evaporate completely. If insoluble material is visible, centrifuge for minutes at,500 x g (5,000 rpm). Transfer supernatant to another 0 ml centrifuge tube and add 60 µl 00% ethanol. Apply the mixture to EZ-0 column that is in a 0 ml Collection Tube. Spin at 8,000 x g (0,000 rpm) for minutes. Discard the flow-through in the collection tube. Add 500 µl of Wash Solution, and spin at 8,000 x g (0,000 rpm) for minute. Repeat Step Discard the flow-through. Spin at 8,000 x g (0,000 rpm) for an additional minute to remove any residual amount of Wash Solution. PRODUCTS ARE INTENDED FOR BASIC SCIENTIFIC RESEARCH ONLY! NOT INTENDED FOR HUMAN OR ANIMAL USE! 0. Place the column into a clean.5 ml Eppendorf tube. Add µl Elution Buffer into the center part of membrane in the column. Incubate at RT for or 3 minutes. Incubating the tube at 37ºC or 50ºC for minutes may increase. Spin at 8,000 x g (0,000 rpm) for minute to elute DNA from the column. 7 8