Hi all, I have a pretty basic troubleshooting question. -- My yields of DNA tend to be very low following phenol/chloroform extraction and EtOH precipitation, and I'm trying to sort out why this may be the case.
For example, I had 7ug of SuperCos (7.9 kB) as determined by Nanodrop. I then digested at one restriction site to linearize the DNA. Then, I extracted DNA using the method below and found I then had only 700ug of DNA by Nanodrop. This has been pretty typical; I always loose most of my material during this extraction. I'd greatly appreciate any tips/hints/etc to increase my DNA yield. I'm following the below protocol:
1. To the aqueous DNA solution, add an equal volume of Tris-HCl (pH 8.0)-saturated phenol:chloroform:isoamyl alcohol (25:24:1). Mix by shaking gently (for solutions of high MW DNA) or vortexing (for low MW DNA).
2. Spin at 13000 rpm for 5 min at 4C.
3. Carefully pipet aqueous layer (top) to a new tube.
4. To this aqueous mixture, add an equal volume of chloroform. Mix as before.
5. Spin at 13000 rpm for 2 min at 4C.
6. Remove aqueous layer (top) to a new tube.
7. Add 1/10 volume of 3M sodium acetate (pH 5.5) to give a final concentration of 0.3M sodium acetate. Mix.
8. Add two volumes of cold 100% EtOH. Mix. Place on ice overnight.
9. Spin at 13000 rpm for 20 min at 4C. Pipet off supernatant carefully.
10. Add 1mL 70% (V/V) RT EtOH to tube with DNA pellet. Vortex or mix gently. Spin 4 minutes at 4C. Pipet off supernatant. Air dry pellet. Resuspend pellet in desired buffer.
Thanks for any and all advice!