Thanks to all those who replied to my query about purification of protein-DNA complex for crystallization. I figured out that extraction from native PAGE is the best way to purify duplex DNA. Does any one have a protocol that had worked for you.
I found the following protocol that I am planning to use. Any ideas and precautions? I am concerned about the chances of Thymine dimer formation upon UV exposure during UV backshadowing. Are there any safer ways of visualizing unstained DNA on gels? Are PD-10 columns (Pharmacia) good for my application? NATIVE PAGE gel - 15% polyacrylamide, , 1x TBE Bio-Rad Protean II gel system 15 x 20cm plates 15% Acrylamide/ /1x TBE 5 well Prep Comb 340 volts 1) Run 4-6 ODs of oligo per well 2) Run gel with Xylene cyanol/Bromophenol Blue marker 3) Run gel until leading dye is about 1 inch above bottom of gel 4) Remove gel to TLC plate (available from VWR; Catalog EM 16484-1) 5) UV back-shadow the gel to visualize the DNA bands. Use transilluminator gel box 6) Cut the correct band out of the acrylamide gel 7) Place the gel piece containing the DNA into a 15ml conical tube 8) Crush gel with pipet (wrap pipet tip in parafilm to keep gel pieces from getting into the pipet.) or autoclaved glass rod 9) Add 3mls HPLC grade Water. 10) Heat at 94*C for 15 minutes 11) Freeze at -70*C for 15 minutes 12) Heat at 94*C for 2 minutes 13) Centrifuge gel pieces to bottom of tube 14) Remove supernatant to a separate 1.5 ml centrifuge tubes (approx 1ml per tube) and Speedvac (lyophilize) down until only about 100ul of liquid remains in the tubes 15) Combine the products into one tube and wash other two tubes with 500ul (total) of water 16) Bring volume total up to 1ml with water 17) Load onto Sephadex G-25 column) 18) Allow liquid to drip through column (by gravity), discard liquid 19) Add 1.5ml water to column 20) Allow liquid to drip through column (by gravity) COLLECT THISÂ…..it has your oligo in it! 21) Measure A260 and calculate amount of oligo in solution (1 OD = 33ugs) 22) Dry oligo in speedvac/lyophilizer 23) Re-suspend oligos to desired concentration. Dept. of Biochemistry, Cellular and Molecular Biology, Walters Life Science, # 406, University of Tennessee, TN, Knoxville, USA
