DNA extraction

This basic extraction protocol works well for both fresh and preserved tissues. It is wise to include a negative control when performing DNA extraction to ensure solution are not contaiminated.

CELL LYSIS SOLUTION: 10mM Tris, 100 mM EDTA, 2% SDS pH 8.0

PROTEIN PRECIPITATION SOLUTION: 7.5 M Ammonium Acetate

TE BUFFER: 10 mM Tris, 0.1 mM EDTA, pH 8.0

NOTE: The TE buffer and Cell Lysis Solution should be autoclaved before use.

  1. If you are using fresh tissue skip to step 2. If using ethanol preserved tissue you must first eliminate as much ethanol as possible. Dissect about 50mg tissue (do not use too much; less can mean more) and place on a clean paper towel and press out the sample a couple of times to remove ethanol. For fin clips or other tissue types not very absorbent this adequate. For absorbent tissue the sample is then placed in a 1.7ml tube with 900 µl TE buffer. Mix well and centrifuge for 1 minute at high speed. Draw off the TE buffer and blot the tissue on a paper towel and proceed to step 2.

  2. Pipette 720 µl of Cell Lysis Solution to a 1.7 ml tube and add tissue sample (about 50mg)

  3. Add 5µl of Proteinase-K (20mg/ml) to each tube. Mix by inversion 10-20 times and incubate at 550C for several hours to overnight (with periodic mixing) until tissue is completely dissolved. Once digested coll to room temprature.

  4.  

 

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