Difference between revisions of "Part:BBa K4436017"

Revision as of 03:55, 12 October 2022

Forward Primer for Lambda Red CsgA Knockout

This is the forward primer for the Lambda Red Knockout of the native csgA in E. Coli.

Usage and Biology

Lambda Red Knockout is a system used by many molecular biologists to knockout single genes or even entire operons (Datsenko and Wanner, 2000). It uses a phage recombinase to replace the native gene in a strain of E. Coli with an antibiotic resistance gene (Datsenko and Wanner, 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 Wanner, 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 Wanner, 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:

Initial Denaturation: 98 C for 30 d

30 cycles 98 C for 10 seconds

66 C for 30s 66 C for 90 s

Final Extension: 72 C at 120s Hold at 4 C

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. Sequence and Features