DNA degradation from phenol-chloroform - how to improve phenol-chloroform extraction (Jan/07/2008 )
I got a issue with phenol-chloroform extraction of DNA from animal tissue (piece of liver)
I did this process so many times without any problem, but these days when I do this extraction,
isolated DNA is almost always degraded.
I have no idea what I am doing wrong.
So, Could you guy teach me tip for this extraction one more time, maybe I need to relearn with the mind of begginer
Any small tips would be fine, things that I need to care, things that I should avoid, anything is appreciated.
The following is the just basic protocol that I follow normally.
Protocol for DNA extraction (reagents/tubes used here are DNAse RNAse free or UV-treated)
1. Use 0.01-0.50 g or fresh tissue and place in a 1.5 ml microcentrifuge tube.
2. Add 1000 ul lysis buffer and macerate tissue.
Lysis Buffer: 10 mM Tris HCl pH 8.5, 5 mM EDTA, 0.2 % SDS, 0.2 M NaCl, 0.1mg/ml Proteinase K
3. Incubate sample at 37C at lease few hours Over night
4. spin for 3 min. at 10,000 rpm.
5. Remove supernatant and place in a new microfuge tube.
6. Add equal volume of buffer-equilibrated phenol-water and invert gently.
7. spin for 10 min. at 10,000 rpm.
8. Remove upper aqueous layer with a pipetter and place in a new microfuge tube.
9. Add equal volume of phenol-chloroform-isoamyly alcohol (25:24:1) and invert gently.
10. Microfuge for 5 min. at 10,000 rpm.
11. Remove upper aqueous layer with a pipetter and place in a new microfuge tube.
12. Add 1/10 volume of 3M Sodium Acetate, and mix well
13. Add 2.5 times of volume of 100% ethanol and invert gently
14. Incubate the solution in -20C freezer O/N
15. Spin the tube at 14000 RPM for 30 min at 4C using tabletop microcentrifuge
16. Wash pellet with 75% ethanol twice
17. Air dry the DNA pellet
18. Resuspend DNA on desired amount of MBG water
Thanks in Advance.
I had the same problem once, and I found out it was due to my proteinase K. I think the proteinase K had lost activity (in my case due to repeated freeze/thawings). The lysis buffer still degraded the tissue, but there must have been active DNases due to the loss of activity of the proteinase K.
So you might try making a new stock of proteinase K, especially if your stock is old or thawed repeatedly.