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Supplementary data: In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

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posted on 31.08.2021, 09:59 by Masayuki Su'estugu, Seia Nara

Supplementary Table: A list of primers used in this study

Supplementary Figure 1. Amplification of a small plasmid contaminant in isothermal RCR. The indicated amount of pRpoABCDZ was subjected into the Tn-oriC insertion reaction, followed by RCR at 30˚C for 16 h.

Supplementary Figure 2. Restriction enzyme analysis of the amplification products of pTT8. Purified pTT8 plasmid or the amplification product of the pTT8 plasmid generated by Tn-RCR were incubated at 37ºC for 3 h with (cut) or without (no cut) KpnI and NheI. The incubated products were then analyzed by 1% agarose-gel electrophoresis and SYBR Green staining. It should be noted that the ratio of supercoiled form decreased in “no cut” sample due to DNA damage during the incubation. Size-marker fragments (M2) were derived from lambda phage DNA. A digestion map of pTT8 is shown on the right.

Supplementary Figure 3. Restriction enzyme analysis of the amplification products of the F plasmid. A purified F-plasmid or the amplification product of the F plasmid generated by Tn-RCR were digested with PmeI as described in Supplementary Figure 2. The digested products were analyzed by 0.75% pulse-field agarose gel electrophoresis using a Pippin pulse power supply (Sage science) and SYBR Green staining. Although this pulse-field methods can separate large sized linear DNA, discrimination of supercoiled DNA band is difficult. Size-marker fragments (M3), Saccharomyces cerevisiae chromosomal DNA. Size-marker fragments (M4) were derived from T7 phage DNA. A digestion map of F plasmid is shown on the right.

Funding

This work is supported by Japan Science and Technology Agency (JST) CREST (JPMJCR18S6 to M.S.), Council for Science, Technology and Innovation (CSTI) ImPACT Program (to M.S.), and Grant-in-Aid for Research Fellow (19J20097 to S.N.) of Japan Society for the Promotion of Science (JSPS). The authors declare the following competing financial interest: M.S. is a co-founder, equity holder and chief scientific officer in OriCiro Genomics, Inc., a company commercializing the large DNA amplification technology used in this study.

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