Genomic DNA Isolation Kit

(Catalog #K281-50; Store at –20^oC)

I. Introduction:

The Genomic DNA Isolation Kit provides a simple and convenient procedure for rapid isolation of genomic DNA from mammalian cells and tissue samples with high yield and purity. The novel method requires less than 90 minutes to prepare highly pure genomic DNA. The extracted genomic DNA is free from protein and RNA, and suitable for a variety of applications such as PCR, DNA hybridization, enzyme manipulation, cloning, Southern blot, and array-based experiments.

1. 
   

   Kit Contents:

   
   

2. 
   

   Genomic DNA Isolation Protocol:

   
   

A. General Consideration and Reagent Preparation:

• 
  

  Read the entire protocol before beginning the procedure.

  
  


• 
  

  After opening the kit, store Enzyme Mix at –70^oC. Store buffers at 4^oC.

  
  


• 
  

  Add 275 μl of TE buffer to Enzyme Mix, mix well, aliquot and refreeze immediately at –70^oC. Stable for up to 3 months at –70^oC.

  
  


• 
  

  Be sure to keep all buffers on ice at all times during the experiment.

  
  


• 
  

  The protocol is designed for using with 1-2 x 10⁶ cells, and generally produces 5-20 μg genomic DNA. If larger amount of DNA is desired, scale up the volumes proportionally.

  
  

1. 
   

   Genomic DNA Isolation Protocol:

   
   

0. 
   

   Collect cells (1-2 x 10⁶) by centrifugation at 600 x g for 5 minutes at 4^oC.

   
   

Note: For tissue samples, ground freshly excised tissue in liquid nitrogen. Weight ~5 mg grounded fine tissue powder in a microcentrifuge tube.

1. 
   

   Add 35 μl of Cell Lysis Buffer. Mix and keep on ice for 1 minute. Vertex for 5 seconds.

   
   


2. 
   

   Centrifuge in a microcentrifuge tube at top speed for 3 minutes . Remove supernatant. The pellet is isolated nuclei.

   
   


3. 
   

   Resuspend the pellet in 40 μl Cell Lysis Buffer.

   
   

4. 
   

   Add 5 μl of Enzyme Mix, pipet several times to mix.

   
   


5. 
   

   Incubate at 50^oC water bath for 1 hour or until the solution becomes clear.

   
   

Note: You may extract the sample using 50 μl of Phenol/Chloroform to remove insoluble materials before doing ethanol precipitation (optional).

6. 
   

   Add 100 μl absolute ethanol, mix and keep at –20^oC for 10 minutes.

   
   


7. 
   

   Centrifuge in a microcentrifuge at top speed for 5 min at room temperature.

   
   


8. 
   

   Remove the supernatant.

   
   


9. 
   

   Wash the DNA pellet 2 times with 1 ml of 70% ethanol. Remove the trace amount ethanol using pipet tip. Air dry for 5 min. (Note: Do not completely dry the DNA. It would be difficult to dissolve if it is completely dried.)

   
   


10. 
    

    Resuspend the DNA in 20 μl TE Buffer or water, store the extracted DNA at –20^oC for future use.