BAC ELUTION AND ION EXCHANGE CHROMATOGRAPHY

Dissolve the polyacrylamide gel:
Cut insert out of ethidium bromide stained BAC gel and weigh the gel fragment in a Falcon tube. The gel slice may be stored in 100 mM NaCl-DEAE buffer at 4°C to avoid drying. To dissolve the polyacrylamide, make the gel (and any buffer it is in) 10% 2-mercaptoethanol (2-ME) in the100 mM NaCl-DEAE buffer. For example, if the gel weight is 2 gm and it is stored in 1.7 ml buffer, add 370 µl 2-ME (i.e. 200 µl for the gel, plus 170 µl for the buffer). Work in the fume hood. Mix by inversion occasionally to dissolve the gel.

Column Preparation and Sample Loading:
Calculate the expected mass of DNA in the gel band (for example, 500 µg of plasmid DNA with a 20% as insert = 100 µg of insert). Add enough DEAE-cellulose (from a 50% slurry) to an autoclaved, disposable Bio-Rad Econo-column or Poly-prep column to bind about twice the expected amount of DNA. The binding capacity of DEAE for DNA is 0.5mg/ml of the packed bed volume. For the above example, load 800 µl of the 50% gel slurry [i.e., 2 (twice the binding needed) x 100 µg DNA x 0.5 mg/ml x 2 (50% slurry)]. Avoid loading the DEAE on the sides of the column because DNA will stick to these places and not be efficiently eluted.

Prepare about 3 ml of a solution of 10% 2-ME in 100mM NaCl-DEAE buffer. Equilibrate the DEAE with this solution by adding about 2.5 ml of this solution to the column. When this has passed through the column, load the DNA, being careful not to disturb the column bed. It may take a long time for the DNA to pass through the column because of the high amount of acrylamide present in thick gels.

Dilute the remaining 10% 2-ME in 100 mM NaCl-DEAE buffer 1:10 to make a 1% 2-ME solution in the same buffer. When the DNA sample has completely passed through the column, wash with 2ml of this 1% 2-ME in 100 mM NaCl-DEAE buffer. This removes the remaining acrylamide and reduces the concentration of 2-ME in the column. Then wash the column with 2ml of 100 mM NaCl-DEAE buffer without 2-ME. This removes the final 2-ME and elutes weakly bound proteins. Finally, add 2ml of 300 mM NaCl-DEAE buffer to elute most of the other proteins.

DNA Elution:
Add 75µl of 1M NaCl-DEAE buffer to the column. Do not collect the first drop or two that falls from the column. You are now ready to collect the sample. Elute the DNA with 100 µl aliquots of 1 M NaCl-DEAE buffer and collect the DNA in a silanized Corex tube. It is a good idea to check how much DNA is still in the column by spotting 0.5µl of the s olution in the bottom of the column on an ethidium bromide plate. Wait about 5 minutes and examine for staining under ultraviolet light. I spot a sample after collecting 600µl, 900µl and 1200µl from the column. Most of the DNA is eluted in the first 600 µl. Dilute the DNA with an equal volume of TE to reduce the salt concentration and ethanol precipitate the DNA by adding 1/10 volume 3M Na acetate plus 2.5 volumes of cold absolute ethanol. < BR>

RECIPES

DEAE-Cellulose Preparation:

We use Whatman DE-52 cellulose (pre-swollen/microgranular). Wash and equilibrate about 50 ml batches as needed in 100 mM NaCl-DEAE buffer. Autoclave 15 minutes.


Ethidium Bromide Stock Solution (10mM Tris-HCl, 1 mM EDTA, 1 mg/ml ethidium bromide)
For 50 ml:
0.5 ml 1M Tris-HCl, pH 8.0 < /DD>
0.1 ml 0.5M EDTA
1 mg ethidium bromide
Add ethidium bromide to Tris-HCl, EDTA and about half of the water, and stir overnight to dissolve. QS to 50 ml with water. Store in a brown bottle at room temperature.


20% Bac-Acrylamide Solution     WARNING: wear gloves
100 ml solution
18.98 g acrylamide
1.02 g BAC (N',N'-bis-acrylylcystamine)
Heat approximatel y 75 ml of water to almost boiling. Add acrylamide and cover with a watch glass. Mix briefly to dissolve and reheat to near boiling. Add BAC. QS to 100 ml with water. Do not autoclave. Filter sterilize with Nalgene 0.45 or 0.2 micron filter and store in foil wrapped bottle.


DEAE Buffers for BAC-Polyacrylamide Gels
100mM NaCl-DEAE Buffer (10mM Tris-HCl, pH 7.9; 1mM EDTA; 0.1 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0. 5 M EDTA
10 ml 5 M NaCl -- or -- 50 ml 1.0 M NaCl
pH to 7.9 and QS to 500 ml with water; autoclave.
300 mM NaCl-DEAE Buffer (10 mM Tris-HCl, pH 7.9; 1 mM EDTA; 0.3 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0.5 M EDTA
30 ml 5 M NaCl
pH to 7.9 and QS to 500 ml with water; autoclave.
1.0 M NaCl-DEAE Buffer(10mM Tris-HCl, pH 7.9; 1mM EDTA; 1.0 M NaCl)
5 ml 1.0 M Tris-HCl
1 ml 0.5 M EDTA
10 0 ml 5M NaCl
pH to 7.9 and QS to 500ml with water; autoclave.


Ethidium Bromide Plates (10mM Tris-HCl, 1 mM EDTA, 0.8% agarose, 2 mg/ml ethidium bromide)
For 1 Liter (approximately 25-30 plates):
10 ml 1M Tris-HCl, pH 8.0
2 ml 0.5M EDTA
8 g agarose
1 L water
Boil to dissolve agarose. Let cool to about 60°C. Add 2 mg ethidium bromide (400µl of 5 mg/ml stock), mix well and pour plates.
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. BAC Elution and Ion Exchange Chromatography. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/bacgels.html). 1997. Oklahoma City. Revision Date: October 2, 1997."