Molecular Biology Protocols

 
Marine DNA Sequencing and Analysis Center
Mount Desert Island Biological Laboratory



 

 

 

 

Last update April 25, 2005. 
Send comments to dtowle@mdibl.org

 

2.  Purification of Total DNA from Animal Tissues

 

QIAGEN DNeasy TISSUE KIT

 

Best results are obtained with fresh material or tissue that has been immediately frozen and stored at -20oC.  To obtain optimum DNA yield and quality, it is important not to overload the process.  For most animal tissues, a maximum of 25 mg fresh tissue can be used in the DNeasy method.  Following proteinase K digestion, tissue samples can be stored in Buffer ATL for up to 6 months at room temperature. 

 

Preparation:  Add indicated amount of ethanol to buffers AW1 and AW2 before using for the first time.  Prepare a 55oC and a 70oC hot block.

 

Procedure:

 

1.  Cut up to 25 mg tissue into small pieces, place in a labeled 1.5-ml microcentrifuge tube, and add 180 μl Buffer ATL.

 

2.  Add 20 μl proteinase K, mix by vortexing for 5 seconds, and incubate at 55oC until the tissue is completely lysed.  Vortex occasionally during incubation or place in a shaking water bath or on a rocking platform in a hot block.

 

3.  Vortex for 15 seconds.  Add 200 μl Buffer AL to the sample, mix thoroughly by vortexing, and incubate at 70oC for 10 minutes.

 

4.  Add 200 μl ethanol to the sample and mix thoroughly by vortexing. 

 

5.  Pipet the mixture from step 4 into a labeled DNeasy Mini Spin Column placed in a 2-ml collection tube (provided).  Centrifuge at 8000 rpm for 1 min in a microcentrifuge.  Discard flow-through and collection tube.

 

6.  Place the DNeasy Mini Spin Column into a second 2-ml collection tube (provided), add 500 μl Buffer AW1, and centrifuge at 8000 rpm for 1 min.  Discard flow-through and collection tube.

 

7.  Place the DNeasy Mini Spin Column into a third 2-ml collection tube (provided), add 500 μl Buffer AW2, and centrifuge at 14,000 rpm for 3 min to dry the DNeasy membrane.  Discard flow-through and collection tube.

 

8.  Place the DNeasy Mini Spin Column into a clean, labeled 1.5-ml microcentrifuge tube (not provided with kit) and pipet 200 μl Buffer AE directly onto the DNeasy membrane.  Incubate at room temperature for 1 min and then centrifuge at 8000 rpm for 1 minute to elute the DNA.  Discard the Mini Spin Column and store the microcentrifuge tube containing the purified DNA at -20oC.