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Lambda DNA Oligo Ligation using Thermo cycler

Genomic DNA from the bacteriophage λ is 48.5 kb long, commercially available (NEB #N3011), and contains 12 nucleotide 5′-ssDNA overhangs that are used to ligate synthetic oligonucleotides.

Contents

Section titled “Contents”
  1. Notes
  2. Procedure

1. Notes

Section titled “1. Notes”

Long DNA is fragile and should not be vortexed. Avoid multiple freeze– thaw cycles, pipette minimally with a wide-hole pipette tip, and mix all solutions gently by inverting and tapping the Eppendorf tube.

Pre-warm the λ-DNA stock at 65–75˚C for a few minutes before pipetting. Pre-warming the λ-DNA melts the cohesive ssDNA ends, which anneal at the high stock concentration.

2. Procedure

Section titled “2. Procedure”
  1. In a 1.5 ml Eppendorf tube, combine 50 μl of 10× NEB Ligase buffer, ~200 μL λ DNA (500 μg/mL), and the desired oligonucleotides.
    1. The oligo concentration should be 2 µM (add 10 μL of stock 100 μM).
    2. Oligos must be 5′-phosphorylated.
    3. If you want to label both ends of λ, both complementary oligos can be added in a single ligation.
    4. We have the biotinylated- and dig-labeled oligos in our communal oligo box.
  2. Add water to bring the total volume to 500 μL.
  3. Gently mix. Do not vortex, as this will shear long DNA
  4. Divide the 500 mL solution into four PCR tubes for the Thermo Cyclers.
  5. Warm to 71˚C in a Thermo Cycler, and let block cool slowly to room temperature by running a program: 71˚C for 15 minutes and then cool down at 0.5˚C/minute (99 times) down to 15˚C.
  6. Once the solution has cooled, add 5 μL of T4 ligase (400 U/μl; NEB #M0202) and place in a RT (room temp) for overnight.
  7. After the ligation reaction is complete, heat inactivate the ligase by heating at 65˚C for 10 minutes.
  8. Filter the reaction through the large Sephacryl S-1000 gel-filtration FPLC column at 4˚C. The running buffer is TE (10 mM Tris 1 mM EDTA) + 150 mM NaCl. Fractionate in 1-ml fractions.
    The column pressure should not exceed 0.2MPa. Run the column at ~0.5 ml/min
    Filtration removes excess oligonucleotide and other reaction components. The 48.5 kb λ-DNA comes out in the void volume of the column. The NaCl prevents non-specific binding of DNA to S-1000 resin.
  9. The dilute λ-DNA fractions collected in 1.5mL tubes are combined in a 15mL or 50mL conical tube and stored at 4˚C.