This is a two step procedure, utilizing a Qiagen Large-Construct Kit for the initial purification, followed by either a chromatographic separation OR gel purification.
Qiagen Prep of BAC DNA
Modified from Biotechniques 27:72-74 (1999)
- Seed 250 ml LB (containing appropriate antibiotic) with approximately 0.5 ml of an over/day culture (containing appropriate antibiotic); grow overnight. Start the large overnight culture at the end of the day and harvest it first thing in the morning--prolonged time at saturation will make it difficult to isolate BAC DNA.
- Spin 15 min at 5500xg to pellet cells; decant supernatant
- Resuspend cell pellet in 50 ml P1 Qiagen buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA)
- Add 50 ml P2 buffer (200 mM NaOH, 1% SDS); invert slowly 4-6 times; incubate 5 min at RT
- Add 50 ml pre-chilled P3 buffer; invert slowly 4-6 times; incubate on ice 30 min
- Spin 60 min at 15000xg at 4°C; filter supernatant through Whatman filter paper
- Equilibrate tip-500 column with 10 ml QBT buffer (750 mM NaCl, 50mM MOPS pH 7.0, 15% isopropanol, 0.15% Triton X-100)
- Apply filtered DNA solution to the column
- Wash column twice with 30 ml QC buffer (1.0 M NaCl, 50 mM MOPS pH 7.0, 15% isopropanol)
- Elute DNA at 65°C with 5x3ml aliquots of QF equilibrated to 65°C
- Add 10.5 ml RT isopropanol to the DNA and mix. Spin 60 min at 15000xg at 4°C; discard supernatant
- Wash pellet with 5 ml RT 70% ethanol and spin 15 min at 15000xg at 4°C; remove supernatant; air dry inverted on paper towel for 15 min. Do not dry the pellet too thoroughly, or it will be impossible to dissolve this large DNA in the next step.
- Add 60 µl of TE buffer to the bottom of the tube; let stand 30 min at RT
- Remove the DNA solution and add 40 µl more of TE to bottom of the tube; let stand 15 min; spin briefly to collect liquid and combine the solutions for total volume of 100 µl
Sepharose Purification of BAC DNA
Modified from Nat Biotech 15:859-864 (1997)
- Use pressurized air to blow the cotton plug to the tip of a 5 ml plastic pipette and clamp the pipette on a stand. Shake CL4b sepharose well and gradually add into the plastic pipette until it is packed almost to top leaving about 1 ml space. Do not let the column dry at any point in preparation of the column or during the chromatography.
- Use a 10 ml syringe to make a reservoir on top of the column (wrapping parafilm around syringe/pipet junction to prevent leaking). Wash the column three times with 10 ml of the injection buffer (autoclave-sterilized 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 100 mM NaCl). This takes 2-3 hours.
- Add 5 µl of any Bromophenol Blue-containing DNA loading buffer to the 0.1 ml of Qiagen-prepped BAC DNA. When the buffer has receded to just above the resin bed, remove the reservoir and add the DNA to the column. Wait until DNA+dye just goes into the column, then add 0.5 ml injection buffer to the top of the column.
- Reattach the syringe-reservoir with parafilm. Add 10 ml injection buffer. Start collecting 0.5 ml fractions with a 24 well plate (about 12 fractions for the dye to reach the bottom of the column). Circular BAC elutes early, about in fractions 4-7, followed by linear BAC and genomic DNA (about fractions 6-9).
- Run 50ul of each fraction on a low percentage agarose gel to identify the early BAC fractions. Typically, the BAC concentration will be 5 to 20 µg/ml. Purified BAC DNA is stable for weeks at 4°C.
Gel Purification Of BAC DNA For Pronuclear Injection
Protocol from Katie Krueger with slight modifications
- Verify correct composition and size of final construct with PCR, sequencing, and pulsefield gel electrophoresis.
- If the insert will be removed from the backbone, digest 10-12 µg of DNA
- Prepare gel box
- Thoroughly wash gel box, tray, and comb (10 well) to remove all traces of ethidium bromide.
- Place gel box with 1x TAE buffer in cold room for several hours to cool.
- Pour 1% gel using low melting point agarose.
- On gel, load the following:
- Lane 1: marker
- Lane 2: 1 µl uncut DNA
- Lane 3: 1 µl cut DNA (if the insert will be purified from the backbone)
- Skip lane 4 and 5
- Lane 6: load rest of digested sample.
- Run gel for 3 hrs at 100 V in cold room.
- Cut the gel down lane 4/5. Stain gel piece with lanes 1-3 with ethidium bromide. Wrap other piece in saran wrap and do not expose to any surfaces that may contain ethidium. Place both sides of gel on the transilluminator. Using the stained half as a reference, cut out the portion of the gel that contains the DNA to be injected.
- Cut about a 1 cm square, cutting no lower than the bottom of the band in the stained side of the gel (to avoid getting any vector DNA).
- Use NEB protocol for agarose digestion.
- Weigh gel slice in microfuge tube.
- Wash gel slice in 1X β-agarase buffer on ice two times for 30 min each.
- Remove buffer, then melt agarose 10 min at 65°C.
- Cool to 42°C (5 min; don't rush, β-agarase is inactivated at temps >45°C!), then add 1 unit β-agarase per 200 µl agarose. Incubate one hour.
- Chill sample for several hours @ 4°C, then check for little chunks of agarose (means digest was incomplete and needs to be repeated!).
Make 50 ml injection buffer (10 mM Tris-HCl, pH 7.5; 0.1 mM EDTA; 100 mM
NaCl). Filter sterilize before using.
Place 50 ml injection buffer in sterile petri dish, then place dialysis discs
(Millipore Cat# VMWP01300, 0.05 µm 13 mm) on top of injection buffer shiny
Drop 50 µl DNA sample on each disc (drop onto center of disc, do not allow to touch the edges!). Cover with petri dish lid, and allow to equilibrate 3-4 hrs on benchtop.
Carefully pipette liquid on top of discs and pool into one tube.
Run 5, 10 (& maybe 15) µl of DNA on a normal gel with marker (1 kb ladder) to determine DNA concentration (expect to be a few ng/µl).