3. Restriction enzyme analysis of the amplification products of the F plasmid.
Table: A list of primers used in this study
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.
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.
A purified F-plasmid
or the amplification product of the F plasmid generated by Tn-RCR were digested
I 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
chromosomal DNA. Size-marker fragments (M4) were derived from T7
phage DNA. A digestion map of F plasmid is shown on the right.
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.