Navigation Links
Genomic DNA Isolation Protocol(1,2,3)

Note: Genomic DNA is fragile. High molecular weight DNA is sheared easily by mechanical forces. Use suitable large-bore pipet tips or equipment when pipetting genomic DNA. Do not vortex solutions containing genomic DNA.

I Isolation of nuclei

Nuclei from blood

Caution: Wear suitable protective apparel and take appropriate safety measures when working with human blood.

  1. Transfer 5 ml whole blood (containing EDTA as the anti-coagulant) to a 15 ml polypropylene centrifuge tube.
    Note: When working with avian blood, or blood from other species that have nucleated red blood cells, use 0.2 ml blood and 4.8 ml 1x Tris-Buffered Saline (TBS, 50 mM Tris-Cl, 200 mM NaCl, 3 mM KCl, 0.02% Sodium Azide, pH 7.5) instead of 5 ml blood.
  2. Add 5 ml 2x Lysis Buffer (0.65 M Sucrose, 20 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 2% Triton X-100) and mix by gentle inversion.
  3. Incubate solution for 5 minutes on ice.
  4. Pellet nuclei by centrifugation at 1000 x g for 12 minutes at 4C.
  5. Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
    Note: The pelleted nuclei may be stored at 70C for several weeks at this point, however, higher molecular weight DNA is obtained when genomic DNA is isolated from freshly prepared nuclei.

    Proceed to Section II.

Nuclei from tissue culture cells
  1. Suspend cells from one 100 mm plate in the growth medium and transfer them to a 15 ml polypropylene centrifuge tube.
  2. Pellet cells by centrifugation at 250 x g for 5 minutes at 4C.
  3. Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
  4. Suspend cells in 1 ml 1x TBS by repeated up and down pipetting, then add 4 ml 1x TBS and 5 ml 2x Lysis Buffer to the cell suspension and mix by inversion.
  5. After a 5 minute incubation on ice, pellet nuclei by centrifugation at 1100 x g for 12 minutes at 4C.
  6. Decant supernatant and drain any residual supernatant by inverting the tube on a paper towel for 2 minutes.
    Note: The nuclei may be stored at 70C for several weeks at this point, however, higher molecular weight DNA is obtained from freshly prepared nuclei.

    Proceed to Section II.

II Lysis of nuclei and deproteinization of DNA
  1. Suspend pelleted nuclei in 2 ml Saline/EDTA Solution (75 mM NaCl, 24 mM EDTA) by repeated up and down pipetting.
  2. Carefully transfer suspended nuclei to a pre-spun (1500 x g for 23 minutes) PLG 15 ml Light tube.
  3. Add 50 L 20 mg/ml Proteinase K (in 10 mM Tris-Cl, pH 8.0, 1.0 mM CaCl2, 30% Glycerol) and 200 l 10% SDS to the suspended nuclei and mix by gentle inversion.
  4. Incubate for 2 hours at 37C with occasional gentle mixing. Note: This incubation may be extended to 1618 hours if that is more convenient.

    Proceed to Section III.

III Extraction of protein
  1. Add 4 ml water-saturated Phenol to the PLG 15 ml Light tube containing the deproteinized DNA. Cap the tube tightly.
  2. Mix by shaking vigorously enough to form a homogeneous suspension. Do not vortex.
  3. Centrifuge at 1500 x g for 5 minutes to isolate the upper, DNA-containing aqueous phase away from the lower, organic solvent phase.
  4. Carefully decant upper phase containing the DNA into a fresh, pre-spun, PLG 15 ml Light tube.
  5. Repeat steps 1 through 3 but this time extracting with 4 ml water-saturated Phenol-Chloroform (PC, 1:1).
  6. Carefully decant upper phase containing the DNA into a clean 15 ml polypropylene centrifuge tube.

    Proceed to Section IV.

IV Precipitation of DNA

At this point, the DNA should have been extracted with both Phenol and Phenol-Chloroform and should be in a clean 15 ml polypropylene screw cap centrifuge tube.

  1. Add 100 l 2 M KCI and mix by gentle inversion.
  2. Overlay DNA solution with 5 ml 95% Ethanol by slowly pipetting the Ethanol down the side of the tube.
  3. Place a Pasteur pipet tip at the interface of the DNA-Ethanol solution and spool the DNA onto the pipet tip by swirling the pipet, keeping the tip at the interface, until the 2 phases are completely mixed.
  4. Place pipet tip with the spooled DNA in 1 ml 70% Ethanol for about 2 minutes.
  5. Remove pipet from the 70% Ethanol and hold upright (tip up) for a few seconds to allow the excess Ethanol to drain away. Do not allow the DNA to dry.
  6. Set pipet tip in a microcentrifuge tube containing 200 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) and incubate for 20 minutes at room temperature. The DNA should slide from the pipet into the TE. If necessary, gently remove the DNA from the pipet by scraping the DNA onto the interior of the tube.
  7. Redissolve DNA completely by incubating overnight at 4C. Store DNA at 4C. Do not freeze.
References
  1. Herrmann, B.G. and Frischauf, A.M. (1987) Isolation of genomic DNA. Methods Enzymol. 152:180-183.
  2. Laws, G.M. and S.P. Adams. (1996) Measurement of 8-OHdG in DNA by HPLC/ECD: The importance of DNA purity. BioTechniques 20:36-38.
  3. Murphy, N.R. and Hellwig, R. J. (1996) Improved nucleic acid organic extraction through use of a unique gel barrier matrix. BioTechniques 21:934-939.

'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
2. New Protocols for Isolating High- Molecular-Weight Genomic DNA
3. Easily Amplify Genomic DNA with Long-Distance PCR
4. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
5. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
6. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
7. Mouse Tail Genomic DNA Isolation Protocol(1)
8. Genomic DNA Isolation Protocol(1,2,3)
9. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
10. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
11. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Genomic DNA Isolation Protocol

(Date:9/16/2014)... cell phones to cars and flashlights, batteries play ... technology companies constantly are seeking ways to improve ... time using a water-based solution, researchers at the ... more efficient nuclear battery that could be used ... source in automobiles and also in complicated applications ...
(Date:9/16/2014)... BlueInGreen® , a water ... for delivering dissolved gases into liquids, announced the ... a manufacturer’s representative firm to its sales network. ... municipal water and wastewater applications in the Canadian ... founded in 1992 and serves Quebec and Ontario ...
(Date:9/16/2014)... MILLBROOK, N.Y. , Sept. 16, 2014  Donald Fresne, ... the appointment of Stephan Vagner , PhD to its ... the University of Toulouse, France , performed ... Germany , and at Inserm, the French institute of ... Inserm and works both at the Curie Institute, Orsay, ...
(Date:9/16/2014)... , Sept. 16, 2014  The Council ... entrepreneurial support organization in the country, today released ... Report . It showed that ... North Carolina startups in the technology, ... more global investment from a variety of sources. Significant ...
Breaking Biology Technology:BlueInGreen® Expands into Canada – Signs Representative Agreement with H2Flow Equipment Inc. 2New Appointment to the Scientific Advisory Committee at Egenix, Inc. 2CED's Mid-Year Update to its Innovators Report Shows NC Startups Attracting Investment From Across the Globe with Several High-Profile Investment Deals, IPOs and Acquisitions Signaling Strength of Entrepreneurial Sector 2CED's Mid-Year Update to its Innovators Report Shows NC Startups Attracting Investment From Across the Globe with Several High-Profile Investment Deals, IPOs and Acquisitions Signaling Strength of Entrepreneurial Sector 3CED's Mid-Year Update to its Innovators Report Shows NC Startups Attracting Investment From Across the Globe with Several High-Profile Investment Deals, IPOs and Acquisitions Signaling Strength of Entrepreneurial Sector 4
... led by Governor Doyle and organized by Forward Wisconsin ... industrys leading conference with goals to establish new partnerships, ... community and to let others know that Wisconsin has ... group, the delegation met its objectives in spite of ...
... Medical Systems, a unit of General Electric Company, announced ... Food and Drug Administration to begin marketing its new ... Signa EXCITE 3.0T combines the increased speed and higher ... of imaging capabilities found on premium 1.5 Tesla systems. ...
... Insurance. Motorcycles? Computerworld has just released their ... 2003, and five Wisconsin companies have placed in the ... Inc. of Milwaukee. , ,This years list, with ... employees perspectives of working conditions and employment benefits. According ...
Cached Biology Technology:On site at BIO 2003 2On site at BIO 2003 3On site at BIO 2003 4On site at BIO 2003 5GE Medical Systems Receives FDA Clearance for New MRI System 2Wisconsin Companies Ranked Top in Nation in IT 2Wisconsin Companies Ranked Top in Nation in IT 3Wisconsin Companies Ranked Top in Nation in IT 4
(Date:9/16/2014)... Spanish . CRG ... the Journal of Cell Biology describes how Topo ... division , At this very moment thousands of our body,s ... which the body repairs damaged tissues and regenerates others like ... as "mitosis", during which the cell duplicates its genetic material ...
(Date:9/16/2014)... vital functions in an organism. For example, they ... and control immune system responses. Researchers established some ... of each other, but instead form complex networks. ... find many similarities with online social networks," says ... Systems Biology. "Some proteins are good networkers that ...
(Date:9/16/2014)... , Sept. 16, 2014 As open ... make important decisions about their 2015 benefits. According to ... health solutions business of Aon plc (NYSE: ... take a more proactive role in managing their health ... than ever for individuals to understand how they can ...
Breaking Biology News(10 mins):Unraveling cell division 2Unraveling cell division 3Good networkers make prime targets 2Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 2Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 3Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 4Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 5Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 6Aon Hewitt Provides Tips to Help Workers Prepare for the Upcoming Open Enrollment Season 7
... , EAST LANSING, Mich. Bacteria infections most of ... world and not malaria are the leading cause of ... new Michigan State University project based in Nigeria and funded ... diseases by collecting local data on the ailments, spurring vaccine ...
... European Molecular Biology Laboratory (EMBL) in Monterotondo, is one ... honoured with a Howard Hughes Medical Institute (HHMI) International ... for five years, is designed to give a boost ... United States. The award recognises the potential of Sotillo,s ...
... concern that excessive levels of fructose may pose a great health ... researchers at the University of Colorado School of Medicine, along with ... if two common sweeteners in western diets differ in their effects ... The study, recently published in the journal Metabolism , took ...
Cached Biology News:$5.8 million Gates grant targets child-killing bacteria diseases in Africa 2CU School of Medicine researchers look at effects of 2 common sweeteners on the body 2
Rad 50 Immunogen: c-terminal mRad 50 (604 amino acids). Storage: 4 C...
...
... Kit Includes: Sufficient library for ... x 10^9 independent sequences, -28 gIII ... DNA sequencing, -96 gIII Sequencing Primer ... Host E. coli strain ER2738, Control ...
... The CHEMICON Re-Blot Western Blot Recycling ... antibodies from Western blots that have ... iodine or other isotopes. It is ... (TMB, DAB, 4-chloronapthol, etc.), as it ...
Biology Products: