Hi.. I am a chemist working on a biochemistry thesis. I have been trying to isolate DNA-aflatoxin adducts from liver tissue using the protocol that was given to me by an instructor from other country. However, we couldn't determine why I wasn't getting any DNA at all. The DNA quality that I need should be suitable for High Performance Liquid Chromatography (HPLC)- meaning it has to be of purest quality.
I was wondering if you could help me determine what is wrong with the protocol. Thank you in advance
Extraction of DNA from Tissue
ref.: Cancer Res., 1986, 46(8), pp. 3924-3931.
a. Tris/Sucrose pH=7.0
for 1 liter: 85.6g sucrose (nuclease free)
1.9 g KCl
2.1 g MgCl2
7.9 g Tris HCl
b.0.5% Triton X-100 in Tris/sucrose
c. 4M NaCl in Tris/sucrose
d. 10% SDS in Tris/sucrose
a) thaw 1 g of liver and homogenize in 4 ml Tris/sucrose using the polytron
b) centrifuge at 1600 rpms (800Xg) for 10 min.
c) discard the supernatant
d) resuspend the pellet in 5 ml Triton X-100 using a Pasteur pipet
e) centrifuge at 2000 rpms for 10 min.
f) discard supernatant and repeat steps d. and e.
g) discard supernatant and add 10 ml Tris/sucrose, 3.8 ml NaCl, and 1.5 ml 10% SDS solution
h) extract with 15 ml CHCl3 –IAA (24:1) by shaking for 15 min.
i) centrifuge 10 min. at 2000 rpms.
j) discard organic layer and repeat h. and i.
k) remove the aqueous layer and precipitate DNA by adding an equal volume of 100% ice-cold ethanol
l) wind out DNA and remove from ethanolic solution; slightly dry
3. Place DNA in an eppendorf tube and dissolve in 1 ml H2O by heating at 37C overnight using the Thermomixer. Remove 250μl and acidify with 50μl 0.1 N HCl. Hydrolyze by heating for 20 min. at 95C in the Thermomixer.
4. Remove 25μl of dissolved DNA from unhydrolyzed tube to quantify the amount of DNA by diphenylamine assay.