Subcloning a fragment from a BAC into a plasmid via gap repair
For subcloning a fragment from a BAC into a plasmid via gap repair, also termed “retrieval”, the
plasmid needed for retrieval can be modified by cloning in adapter oligos.
1. Design top and bottom strand oligos so that they can anneal together to give a doublestranded oligo with the following properties:
• will have “sticky end” overhangs to ligate to restriction sites in the destination plasmid
• will have the left and right side homology arms in the correct order and orientation
• between the homology arms, leave a unique restriction site that does not cut elsewhere
in the destination vector. It does not matter if this site is present in the subcloned fragment or
not.
Thus the structure of the annealed, double stranded oligo should be:
Sticky end for site “A” – Left arm – Unique site “C” – Right arm – Sticky end for site “B”.
When ordering the oligos, request the scale and purification recommended by the vendor for
oligos of that size.
2. Anneal the oligos:
See Annealing protocol.
3. Cut destination plasmid with enzymes A and B. Gel purify the linear plasmid and quantify.
4. Perform ligation with the vector and annealed ds-oligo. Suggested method: NEB quick
ligation kit with only 2.5 ng/µl plasmid and 0.5 ng/µl adapter in total 20 µl volume as specified by
the kit. Transform the ligate, and screen resulting colonies for clones that have a plasmid that
now cuts with the unique site C. If desired sequence the plasmid to verify cloning of the insert.
5. To prepare the plasmid for gap repair recombination, digest fully with enzyme C and carefully
gel-purify the linear band. Care is required to minimize contamination with uncut plasmid,
which can cause background at the recombination step. You will need >200 ng of purified DNA
in < 5 ul of pure water for the recombination step.
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Doug Mortlock 2011
Annealing oligos
ADB (Adapter dilution buffer): 50 mM NaCl / 10 mM Tris pH 7.5
10X Annealing Buffer: 1 M NaCl / 10 mM EDTA pH 8.0 / 100 mM Tris pH 7.5
1. Resuspend oligos in water to approximately 1 µg/µl concentration. (The actual total µg’s of oligo is
usually supplied by the oligo manufacturer on the spec sheet or on the tube).
2. Make up annealing reaction with 20 µg each oligo in total of 400 µl 1X annealing buffer, using 10X
annealing buffer stock and water.
3. Heat several hundred ml of water in a beaker till just boiling. Remove from heat. Float the annealing
reaction in the beaker. Let cool to room temperature on the benchtop.
Alternative to step 3: Transfer the 400 µl oligo mix to 4 PCR tubes. Cycle in thermal cycler with
a program with initial step of 100˚ for 5 minutes, then ramp the temperature down by 1 degree per minute
for 75 minutes, so the final temp should be about 25 degrees (room temp).
4. Centrifuge briefly. The final concentration of double-stranded, annealed oligo should be close to 100
ng / µl. It can be assumed that most of the oligos have annealed. Dilute in ADB so that an appropriate
amount can be removed for ligations (usually around 1 ng, see notes below)
5. (Optional): To precipitate, add 40 µl 3M sodium acetate, pH 5.2 and 1.0 ml ethanol. Inc. at -20˚ for > 30
min. Centrifuge 15 min., remove sup., add 100 µl 70% ethanol, respin 5 min., remove sup. and let air-dry
5 min. Resuspend annealed oligos in ADB. Determine concentration by UV spec.
Notes:
1. Short annealing sequences may un-anneal in low salt buffers or water. Therefore storage or dilution in
50 mM NaCl (such as ADB) is advised.
Tips for ligating ds-oligos/adapters to cut plasmids:
2. Excessive molar ratios of adapters can inhibit recovery of desired ligation products. Remember to
adjust adapter concentration in downstream ligations to have a good adapter/plasmid molar ratio; i.e. 20:1
or 10:1 is fine.
3. Most adapters will be around 20-110 base pairs long. A typical ligation should work well with 25 ng/µl
plasmid and 1.0 ng/µl adapter. Relatively few ng of adapter is required because they are so small. I use
the NEB quick ligation kit with only 2.5 ng/µl plasmid and 0.5 ng/µl adapter and have excellent results.
4. Remember that synthesized oligos do not have phosphorylated 5' ends, so they will not ligate to
dephosphorylated plasmids (i.e. linearized plasmids should NOT be pre-treated with CIAP or SAP).
5. If you cut your plasmid with two enzymes at adjacent sites, (e.g. to insert an adapter) gel-purification
of the linear plasmid is important first for getting rid of the small internal “stuffer” fragment. This
fragment could otherwise inhibit adapter ligation by re-ligating back to the vector. We have found this
really helps for good efficiency.
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