Michael Court
Updated March 2003
gDNA prep from liver tissue by DNAzol for PCR
1.
Wear gloves to avoid Dnase contamination!!!
2.
Wash out Wheaton 10 mL homogenizer with Rnase Out (peroxide/detergent)
a. This removes contaminating DNA and Dnases
b. Add about 0.5 to 1 mL Rnase Out, mix up and down, and rinse thoroughly
with distilled water
c. Final rinse with 100% ethanol and let dry
d. Keep pestle from touching anything (DNA/Dnase contamination)
3.
Weigh out 25-50 mg frozen liver tissue in weigh boat
a. Use new weigh boat and wash with 100% ethanol and allow to dry
b. Tare balance with empty weigh boat
c. Don’t allow tissue to thaw
d. If necessary cut off needed amount of tissue using (new) single edged
razor rinsed with 100% ethanol and dried
e. If not using tissue immediately, keep frozen (freezer or on dry ice)
4.
Homogenize tissue completely
a. Transfer tissue to Wheaton homogenizer (cleaned as above)
b. Add 1 mL DNAzol
c. Use pestle to completely homogenize tissue using up and down and
twisting motion (about 1 minute; 15-20 strokes)
d. Try to minimize amount of homogenization to minimize DNA shearing
e. DNA is stable at room temperature once homogenized
5.
Remove insoluble material
a. Transfer to 1.5 mL Rnase/Dnase-free Eppendorf tube
b. Centrifuge at 10,000 x g for 10 min at room temperature
c. Transfer supernatant to new Eppendorf
6.
Precipitate DNA
a. Add 0.5 mL 100% ethanol (0.5 volume)
b. Mix by inverting tube 3-6 times
c. Sit on bench for 1-3 minutes
d. Centrifuge at 10,000 x g for 10 minutes at room temperature
e. Pipette off and discard supernatant
7.
Wash DNA pellet twice
a. Add 1 mL of 75% ethanol (in Rnase free water)
b. Mix by inversion
c. Centrifuge at 4,000 x g for 5 min
d. Discard supernatant (remove as much ethanol as possible)
e. Repeat
8.
Dissolve DNA in water
a. Air dry pellet with cap open for 5 – 10 minutes
b. Do not allow to completely dry out or DNA will not dissolve
c. Add 200 uL 8 mM NaOH in Rnase-free water
i. Make up NaOH/water < 1 month ago from concentrated stock
ii. Alkaline solution aids DNA dissolution
d. Allow time for DNA to dissolve
i. Can take several hours at room temperature
ii. Mix by vortexing if don’t need high MW DNA, or flick tube
iii. Best to leave overnight at 40C
iv. Heating to 600C for 10-15 minutes can also help dissolution
e. Once dissolved, buffer to pH 7 by adding 20 uL 1 M HEPES buffer (or
200 uL 0.1 M HEPES)
f. Residual ethanol should be evaporated at this point by heating the opencapped tubes to 600C for 10 – 15 minutes
9.
Measure DNA concentration by fluorescence dye method (Yo-Pro)
10.
Dilute a portion of this to 0.1 ug / uL for PCR work (200 – 300 uL final
volume)
11.
Store at –800C