Sepharose 4B-CL Chromatography BAC DNA Purification for Transgenic Production

This method was contributed by Wes Dunnick  at the University of Michigan Medical School in Ann Arbor, Michigan. The approach is to prepare the DNA with as little manipulation as possible, so that the BAC insert will remain essentially intact, without a significant amount of sheared fragments.  The resulting preparation has both RNA and protein contamination, which probably affects (to a small extent) digestion by restriction enzymes.  The preparation is cleaned up substantially by the final  Sepharose 4B-CL chromatography step.

This protocol is essentially an adaptation of a miniprep from David Kohrman’s laboratory, with the added chromatography step mentioned in Yang, Model, and Heintz (Nature Biotechnology 15: 859-865, 1997).  We have used the Heintz method for mutation of BACs with great success.

1. We maintain our BACs as streaks on bacterial plates or as competent cells frozen at -80C.  If the bacteria containing a particular BAC die, that BAC is probably lost forever; getting enough intact DNA together for transformation of new bacteria would be very difficult.

2. Grow two 50 ml cultures of the BAC in 12.5 micrograms/ml chloramphenicol (the resistance marker carried by our particular BAC), starting each from a single colony and growing overnight at 37C.

3. Harvest each culture by centrifugation at 4000 rpm in a Sorvall SA-600 rotor at 4C.  We treat the samples from this point with the precautions normally associated with RNA preparation.  We use autoclaved tubes and reagents, and wear gloves, since a small amount of DNase would result in many of the BAC molecules being cut once or more.  Hence, we harvest in 30 ml siliconized and autoclaved Corex tubes.

4. Pour off the bacterial culture media, and place the tubes on ice so that the remaining fluid can drain away from the bacterial pellet.  After a few minutes, remove the last of the fluid with a Pasteur pipet. Suspend and combine the cell pellets from each 50 ml culture into a total of 4 mls of 50 mM Tris (pH 8.0), 10 mM EDTA with 400 micrograms/ml RNase A.  Make sure you have a single cell suspension.  Clumps of bacteria will not lyse well and will not result in a high yield of BAC DNA.  We use a Pasteur pipet to both "rub" the cell pellet on the side of the Corex tube and to suspend the cells by repeated aspiration.

5. Immediately add to each combined cell pellet from a 50 ml culture 4 mls of 0.2 M sodium hydroxide, 1% SDS.  We make this fresh from a pellet of sodium hydroxide, 5% SDS, and an appropriate amount of sterile water (usually about 16 ml for one sodium hydroxide pellet).  To lyse the bacteria, cover the tube with parafilm and rotate slowly.  The solution should become translucent, without clumps of bacteria.  Hold at room temperature for 5 minutes.

6. Add dropwise to each combined cell lysate (from a 50 ml culture) 4 mls of 3 M potassium acetate, pH 5.5.  As you add the potassium acetate, shake the tube gently to mix.  Ideally, a genomic DNA:SDS:protein  precipitate will form, and the remaining solution will be completely clear.  Place on ice 15 minutes.

7. Spin at 7500 rpm in the SA-600 rotor for 15 min at 4C.  Remove supernatant, avoiding the small pieces of precipitate which inevitably float in the supernatant.  Add the supernatant to 7 ml of 100% isopropanol at room temperature.

8. Immediately spin at 7500 rpm in the SA-600 rotor for 15 min at 4C.  We have obtained larger pellets by using more isopropanol, holding the sample at -20C before centrifugation, etc.  However, almost all of  the larger pellet is RNA and protein, which inhibit subsequent restriction enzyme digestions.  You may lose 20 to 50% of the BAC DNA using this precipitation approach, but what you have will be high quality.

9. Wash the pellet twice with 5 mls 70% ethanol.  Recover each time by centrifugation in the SA-600 rotor at 7500 rpm for 5 min at 4C.

10. Remove as much of the ethanol supernatant as possible, and allow to dry until no ethanol microdrops are visible, but the nucleic acid pellet may still be shiny (30 to 60 min).  Cover each pellet (originally from a 50 ml culture) with 0.15 ml of 10 mM NaCl.   Allow the pellet to dissolve by just sitting at room temperature for 30-60 min.  If you add higher concentrations of NaCl or spermine/spermidine at this point, the high molecular weight BAC DNA will not dissolve.

11. Shake the solution very gently to finish the dissolving process.  Add 1.5 microliters of a solution of 3 mM spermine/7 mM spermidine.  Spermine and spermidine allow the BAC DNA to assume a "ball-like" structure that is much more resistant to mechanical damage, but is able to be cut by restriction enzymes.

12.  (Usually next day)  Digest 15 microliters with NotI in a 40 microliter volume at 37C for 4 hours or so.  (Not1 releases the insert from the vector for our particular BAC.)  We use yellow tips with wide openings to dispense the BAC.  We find that we can vortex the digestion solution once to mix all the ingredients without shearing the BAC DNA.  Fractionate the digest on a CHEF gel, with appropriate size markers and DNA concentration standards.  We usually find that 15 microliters is about 2 micrograms of BAC DNA, and that the digestion goes to completion.  This should be verified by a Southern blot of the CHEF gel (use a standard Southern blot protocol, but pretreat the gel for 15 min with 0.1 M HCl to allow the BAC insert to transfer better.)  We hybridize with a probe for the CAT gene, which should hybridize to the BAC vector at 11 kb, but not to the insert.

13. We test the DNA from each 50 ml culture separately.  There is some variation in preparations, so one aliquot might have more BAC DNA, or digest to completion more efficiently.  In the end, the two preparations are usually very similar, and we pool them.

14. If the digestion appears to be complete, scale it up to digest 0.15 to 0.3 ml of the BAC DNA.  The digestion solution should include 30 microM spermine and 70 microM  spermidine.  After the digest is complete, apply it directly to a Sepharose 4B-CL column which has been equilibrated in 0.1 M NaCl, 10 mM Tris (pH 7.5), 0.25 mM EDTA (injection buffer).  We pour the column in a 2 ml, siliconized glass pipet, with the end broken off a little so that it has a larger bore.  We attach the barrel of a ten ml disposable syringe to the top of the pipet as a buffer reservoir. Elute the BAC insert using the same buffer, collecting about twelve 0.3 ml fractions. .  (We do this by hand; it takes 1-2 hours).  We add 3 microliters of the 3 mM spermine/7 mM spermidine solution to each tube before fractionation, so that the BAC will be in the appropriate solution immediately as it exits the column. Store the BAC DNA at 4C.

15. Apply 35 microliters from each fraction to a CHEF gel, with appropriate size markers and DNA concentrations standards.  We find that the vector elutes in fractions 3 or 4, and the BAC insert (230 kb in our case) elutes in fractions 6 or 7.  (Fig. 1--This is counter intuitive, but it works this way every time.)  The absorbance of fractions 9-11 are very high (sixty times higher than fractions 6 or 7), suggesting that the RNA elutes late, as it should on a gel filtration column.

16. Fraction 6 or 7 have the BAC insert at about 30 micrograms per ml, and hence can be diluted about 60-fold (without further manipulation) for injection into eggs.  Quantitation: Check concentration by 0D 260/280.  When you submit the BAC DNA for microinjection, also submit 10 microliters of Not I digested BAC DNA for pulsed field analysis. This will allow us to assess the purity of your DNA preparation. We will verify the concentration of your concentrated DNA sample and adjust it for microinjection to 0.5 to 1.0 ng per ul.

Our latest preparation yielded 74 pups from two days of injection.  We screened 70, and found 12 founders.  Our subsequent Southern analysis of transgenic DNA indicates that most founders have DNA from the 5’ portion and the 3’ portion of the BAC in approximately equal copy numbers, suggesting that most copies of the BAC entered genomic DNA intact.



Figure 1:  Not1-digested ARS/Igh BAC was applied to a Sepharose 4B-CL column.  35 microliters of fractions 2-11 were fractionated on a CHEF gel (programmed for fractionation from 20 to 400 kb).  Left portion: Ethidium bromide stained gel.  The lanes with fractions 2 to 8 are labeled.  The right-most lanes contain phage lambda DNA, as indicated.  Right portion: Southern hybridization results, using a probe to the CAT gene.  All of the CAT-hybridizing material is in the vector in lanes 3 and 4; the insert (lane 6 and 7) is virtually devoid of vector.

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