Modifications to BAC DNA Maxiprep procedure using Nucleobond BAC 100 columns.
Doug Mortlock, 2011. Adapted directly from the Nucleobond BAC 100 user manual; my modifications
are highlighted.
1
Cultivate and harvest bacterial cells
Inoculate the BAC cell stock into 250 ml LB + antibiotics for overnight culture. Shake overnight
at the appropriate temperature.
Harvest bacteria from the LB culture by centrifugation at 4,500–6,000 x g for 15 min at 4 °C.
2
Cell lysis
Carefully resuspend the pellet of bacterial cells in 36 mls of Buffer S1 + RNase A. It is critical to
completely resuspend the cells.
Add 36 mls of Buffer S2 to the suspension. Mix gently by inverting the tube 6–8 times.
Incubate the mixture at room temperature (18–25 °C) for 2–3 min (max. 5 min). Do not vortex, as this
will release contaminating chromosomal DNA from cellular debris into the suspension.
Add 36 mls pre-cooled BufferS3 (4°C) to the suspension. Immediately mix the lysate gently
by inverting the flask 6–8 times until a homogeneous suspension containing an off-white flocculate is
formed. Incubate the suspension on ice for 5 min.
3
Equilibration of the column
Equilibrate a NucleoBond® AX 500 (Maxi), BAC 100 (Maxi), or AX 2000 (Mega) Column with 6 mls of
Buffer N2. Allow the column to empty by gravity flow. Discard flow- through.
4
Clarification of the lysate
Clear the bacterial lysate by following centrifugation AND filtering in succession. (Note, the
Nucleobond manual says to use either option 1 or option 2; this protocol has both). This step is
extremely important; excess precipitate left in suspension may clog the NucleoBond® Column in later
steps.
4a. Centrifuge the lysate at >12,000 g for 30 minutes at 4 degrees. ( = 8500 rpm in Mortlock
lab fiberlite F145 rotor). After the spin, the lysate should be extremely clear.
4b. Filter the lysate through a Nucleobond folded filter placed in a funnel in a collecting tube.
5
Binding
Load the cleared lysate from step 4 onto the NucleoBond® Column. Allow the column to empty by
gravity flow.
6
Washing
Wash the column twice with 18 mls Buffer N3. Repeat as indicated. Discard flow-through.
7
Elution
Elute the plasmid DNA with 15 mls Buffer N5.
We recommend precipitating the eluate as soon as possible (step 8). Nevertheless, the eluate can be
stored in closed vials on ice for several hours. In this case the eluate should be preheated to room
temperature before the plasmid DNA is precipitated.
8
Precipitation
Add 11 mls of room-temperature isopropanol to precipitate the eluted plasmid DNA. Mix carefully,
mark the side of the tube that will face outward during the centrifuge step to note where the pellet will
be, and centrifuge at ≥ 15,000 x g for 30 min at 4 °C. Carefully discard the supernatant. Do not be
concerned if the pellet is not clearly visible.
9
Wash and dry DNA pellet
Add 5 mls of room-temperature 70 % ethanol to the pellet. Vortex briefly and centrifuge at ≥ 15,000
x g for 10 min at room temperature (18–25 °C).
Carefully remove ethanol from the tube with a pipette tip. Allow the pellet to dry at room temperature
(18–25 °C) between 5-15 min.
Over-drying may render the DNA less soluble.
10 Reconstitute BAC DNA
Dissolve pellet by adding 200 µl of buffered TE to the presumed location of the pellet. Gently swirl the
liquid around the bottom of the tube to allow it a chance to start hydrating dispersed pieces of the
DNA pellet. Place the centrifugation tube on a platform shaker or 3D-shaker with slow, gentle
agitation for 10-60 min. at 4˚ or room temperature.
Remove the BAC DNA with a wide-bore pipette tip. Transfer to a screw-cap microcentrifuge tube and
store at 4˚, not in the freezer.
Determine BAC yield by UV spectrophotometry. Typically, we recover 100-200 ng of purified BAC
DNA. The 260/280 absorbance ratio should be approx. 1.85, and the 260/230 absorbance ratio
should be greater than 2.0. It is also critical to check that the DNA really is BAC DNA by agarose
gel analysis of a restriction digest.
DPM 12/1/11
Major changes I made as compared to the vendor manual:
1. 50% reduction in culture volume (250 ml, not 500)
2. 50% increase in lysis buffer volumes
3. Both centrifugation and filtration are used to clear the lysate.
The most common problems encountered with the standard manufacturer’s protocol are clogging of the column,
or very little yield of DNA. These modifications greatly reduce the likelihood of these problems at the expense of
some DNA yield.
Because the amounts of buffers S1, S2 and S3 are increased, to use all 10 columns in a kit you will need to
make extra amounts of these buffers yourself; alternatively you can order an extra buffer set.
Kit ordering information:
Nucleobond BAC 100 column kit: Clontech is distributor, catalog # 740579 /Macherey-Nagel is
manufacturer)
Nucleobond extra buffer kit: Clontech catalog # 740601.