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 THIS…..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
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