Recombineering Lambda Phage (Hongshan's Version)

This protocol is combination of the protocol of Recombineering Lambda Phage (Jim’s version), Yoori’s scientific reports paper (2017) and my experience.

pKD78 plasmid (pIF:284, chloramphenicol resistant) harbors the Red recombineering system under the control of an arabinose-inducible promoter.

Contents

1. Preparing the Insert
2. Recombineering
3. References

1. Preparing the Insert

The insert is designed and directly ordered as gblock from IDT, which will greatly increase the recombineering efficiency.

2. Recombineering

Purpose: Lysogens containing pKD78 was transformed with insertion cassette. Fresh electrocompetent cells were prepared for every recombineering reaction. A 3 mL LB culture of strain IF189 (λ-lysogen) transformed with pKD78 was grown overnight at 30 °C in the presence of 30 μg mL−1 chloramphenicol. The following day, 1.2 ml of cells were used to inoculate a fresh 120 mL culture of LB containing the same concentration of antibiotic. When the cells reached an O.D.600 ~ 0.5, the Red recombinase system was induced by adding 2% L-arabinose (Sigma-Aldrich A3256) (Control (uninduced cells): One control without L-arabinose should be considered. Take 40 mL from 120 mL culture before adding L-arabinose.) and incubated for an additional 2 hour at 30 °C. Cells were harvested at 4,500 RCF for 7 min, washed three times in ice-cold Milli-Q H2O, and finally resuspended in 400 µL of H2O (For uninduced cells control, resuspend in 200 ul of H2O.). Electrocompetent cells were kept on ice and used immediately for the recombineering reaction.

For recombineering, 50-150 ng of targeting DNA (pIF 743, pIF 744 for site1, pIF 745, pIF 746 for site2) (I tried purified PCR-ed gBlock and non-PRC-ed gblock. The non-PCR-ed gBlock works. Control (induced cell without DNA): One control without targeting DNA should be considered. 200 ul cells were performed electroporation without adding targeting DNA.) was electroporated with 200 µL cells at a voltage of 2.5 kV (12.5 kV cm−1) in 0.2 cm cuvettes using a micropulser (Bio rad #165-210). Cells were immediately resuspended in 1 mL of SOC and then transferred to 15 mL Falcon culture tubes. After at least 6 hours (I tried 3 hours, 6 hours, overnight. 6 hours and overnight work for me.) outgrowth at 30 °C, 350 µL of the culture was plated onto LB agar plates containing either 25 µg mL−1 carbenicillin or 25 µg mL−1 kanamycin (50 µg mL−1 carbenicillin also works good. 50 µg mL−1 kanamycin did not work for me.) Colonies grow slowly on LB agar plates. It may need 2 overnights incubation at 30 C. Colonies were checked for successful incorporation of recombinant DNA via colony PCR with oligos 751 and 752 for site 1, 753 and 754 for site 2.

3. References

1. Yoori Kim, Armando de la Torre, Andrew A. Leal & Ilya J. Finkelstein. Efficient modification of λ-DNA substrates for single-molecule studies Scientific reports 7, 2071 (2017).