Genomic DNA isolation and PCR amplification

Genomic DNA was extracted from an overnight culture of E. coli K12 MG1655, using the Wizard® Genomic DNA Purification Kit (Promega). The kit can be used for at least 100 isolations. One extraction yields up to 100 µg of genomic DNA (from 5 ml overnight culture) which can be used for several hundreds of amplification reactions. Isolated genomic DNA was subsequently stored at 4 °C. Two means of reducing the cost of DNA extraction are to either manually extract the DNA or fully automate the process. The former approach can lead to a very high yield of DNA at a small cost, and all used buffers and reagents can be made in-house. For more information, see https://bio-protocol.org/bio101/e97#biaoti1286.

DNA amplification was performed via PCR using the Q5 DNA polymerase and 5× Q5 buffer (New England Biolabs) in 50 µl. All primers purchased from Integrated DNA Technologies (IDT). In all PCR reactions, 10–50 ng of E. coli genomic DNA and 25 pmol of forward and reverse primers were used. The PCR protocol consists of: (1) 3 min at 98 °C, (2) 20 s at 98 °C, (3) 20 s at 62 °C, (4) 15 s at 72 °C, (5) go to step 2 and repeat the cycle 36 times, (6) 8 min at 72 °C. Each PCR reaction produced ~2–2.5 µg of the register string, sufficient for >100 reactions. PCR products were run on 1% agarose gel and purified using the Zymoclean gel DNA recovery kit (Zymo Research). Supplementary Table ⁴ lists all the PCR products used in this work. Supplementary Table ⁵ contains the primers used for amplifying these sequences.