EcoRI restriction enzyme

Protocol tips

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	The 5′ PpAOS1 genomic fragment was digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) and inserted into pTN182, which carries a G418-resistant cassette, digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) to obtain pTN182-PpAOS1KO5′.

Protocol tips
The 5′ PpAOS1 genomic fragment was digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) and inserted into pTN182, which carries a G418-resistant cassette, digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) to obtain pTN182-PpAOS1KO5′.

Publication protocol

To construct a vector for PpAOS1 gene disruption, a 1.0-kb genomic DNA fragment beginning 5′ to PpAOS1 was amplified using KOD FX DNA polymerase (Toyobo, Osaka, Japan), and the primers PpAOS1KO5′-F and PpAOS1KO5′-R. A 1.0-kb genomic DNA fragment ending 3′ to PpAOS1 was also amplified using primers PpAOS1KO3′-F and PpAOS1KO3′-R. Each fragment was cloned into the vector pBluescript SKII (+) (Merck, Darmstadt, Germany). The 5′ PpAOS1 genomic fragment was digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) and inserted into pTN182, which carries a G418-resistant cassette, digested with XbaI (Takara Bio Inc., Shiga, Japan) and EcoRI (Takara Bio Inc., Shiga, Japan) to obtain pTN182-PpAOS1KO5′. Similarly, the 3′ PpAOS1 genomic fragment was inserted into pTN182-PpAOS1KO5′, which had been digested with SphI (Takara Bio Inc., Shiga, Japan) and NdeI, (Takara Bio Inc., Shiga, Japan) to yield pTN182-PpAOS1KO.

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