{GENE PREP}

I.       Purpose:    To extract and purify genomic DNA from mouse tail and other types of animal tissues.

II.    Chemical Principal:  Proteinase K/organic extraction method.

IIIA. Preparation of Tissue Digest (manual ProK digest for mouse tail):

A.    Cut 5 - 10 mm of tail (about 10 - 20 mg) and place it into AutoGen Tube Unit.

B.    Add 250µl of a buffered Proteinase K solution (250µl of Reagent GPR-1 including 0.4mg/ml Proteinase K and 250µl of Reagent GPR-2).

C.    Incubate at 55°C - 60°C for 6 hours to overnight, and load tube units onto the GENE PREP.

IIIB. Preparation of Tissue Digest (on-board Pro K digestion for mouse tail):

A.    Cut 5 - 8 mm of tail (about 10 mg) and place it into AutoGen Tube Unit.

B.    Load AutoGen Tube Unit onto the GENE PREP.

IV. Protocol Parameters:

A.    Sample Volume: 0.5ml of digest solution.

B.    Maximum Number of Samples: 48 samples per run
                                                     192 samples per run (with an optional Tube Unit Stacker)

C.    Processing Time: 2.1 hours for 48 samples (if digest is prepared manually).
                              5.5 hours for 48 samples (if digestion is done on GENE PREP)
                              (Includes 40 min drying and re-hydration time).

D.  Yield: 1-2 mg of DNA per 1 mg of mouse tail (manual digestion).
         0.5-1 mg of DNA per 1 mg of mouse tail (on-board digestion).

E.     Quality:    Typical OD_260/280 values are ³ 1.8
             The DNA is RNA free and can be used directly in downstream processes such as
                fluorescence DNA sequencing, PCR, Southern blotting* and restriction endonuclease digestions.
                *If the tissue is digested on GENE PREP, the OD260/280 is lower (around 1.7) and the DNA may not be
                  suitable for Southern blotting analysis.

V.     Running the Protocol:

       A.    Load Reagents and Samples
         B.    Select a Protocol
         C.    Enter Number of Samples
         D.     Start the Run

VI. Example of Data:

   Genomic DNA obtained from mouse tail 

   Stating Sample: 5-10 mm mouse tail (about 10-20 mg)
   Digestion: 55°C for 14hrs manually
   Gel Condition: 5 ml of 100ul resuspended DNA
                         1% agarose gel, 100V x 1hr
 

   PCR of mouse tail DNA 

   Template: 200ng of the above DNA
   Target Region: ICAM-1 (535bp)
   PCR Reaction: 35cycles
   Gel Condition: 10 ml of 25ul PCR reaction                        
                              1% agarose gel, 100V x 1hr                           
                              øX174 Hinc II size marker                        
                              C+/C-: positive and negative controls 

VI.    Extraction and Purification Process: