Part:BBa_K4436011

Usage and Biology

Lambda Red Knockout is a system used by many molecular biologists to knockout single genes or even entire operons (Datsenko and Werner, 2000). It uses a phage recombinase to replace the native gene in a strain of E. Coli with an antibiotic resistance gene (Datsenko and Werner, 2000). One of the first few steps of a Lambda Red Knockout is the PCR amplification of a custom primer, with homology to both the antibiotic resistance gene (in the recombinase sites next to it) and the native gene (Datsenko and Werner, 2000). Accordingly, this primer can be used to amplify the antibiotic resistance gene while allowing for the recombinase to recognize this site and replace the native gene with said gene (Datsenko and Werner, 2000).

Characterization

We begin by preparing a PCR mix on ice, which consisted of: 32.5 microliters of Water 10 microliters of Q5 Reaction Buffer 2.5 Microliters of the Forward Primer 2.5 Microliters of the Reverse Primer 1 microliter of 0.1ng/microliter PKD3 0.5 Microliters of Q5 DNA Polymerase.

We then ran the following PCR Program: 1. 30 seconds of Initial Denaturation at 98 C 2. 30 cycles - 98 C for 10 s - 66 C for 30 s - 66 C for 30s/kb 3. Final ExtensionL: 72 C for 120 seconds 4. Hold at 4 Seconds

Finally, we then ran the results on a 1% Agarose Gel to confirm the results. We were expecting a band at 1.1 kB, which was about the size of the pKD3 plasmid.

As you can see below, we obtained those results around the first month of August.

References

Datsenko, Kirill A., and Barry L. Wanner. “One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products.” Proceedings of the National Academy of Sciences, vol. 97, no. 12, 2000, pp. 6640–6645., https://doi.org/10.1073/pnas.120163297.