Sybr safe nucleic acid stain

Genomic DNA was extracted from rice leaves of the low amylose varieties from Cambodia, Thailand, Australian, and Japan using the method described previously [28] (link). Leaf samples were frozen in liquid nitrogen and ground into fine powder prior to addition of extraction buffer. PCR amplification was performed using a G-Storm Thermal Cycler (model GS1, Gene Technologies Ltd, Essex, UK) in a 20-µL reaction volume containing forward and reverse primers (Table 2), designed with Primer 3 software [29] , and components from KAPA HiFi HotStart PCR Reagent Kit (KAPA Biosystems, Boston, Massachusetts, USA). Twenty microliters of the PCR product were electrophoresed through a 1.2% agarose gel, stained with SybrSafe nucleic acid stain (Invitrogen, Carlsbad, CA, USA), and visualized using a non-ultraviolet transilluminator (Dark Reader DR195M, Clare Chemicals, Dolores, CO, USA). PCR fragments from agarose gels were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. Sequencing of the PCR products was done by Macrogen Inc., Seoul, South Korea. Nucleotide sequences were retrieved and aligned using BIOEDIT software [30] with the Nipponbare rice Wx gene sequence from GRAMENE [31] (link) as the reference.

Triplicate HB27 and HB27Δago cultures with or without plasmid pMHPnqosGFP were cultivated in medium with and without antibiotics to an OD_{600 nm} of 0.5, after which genomic DNA was purified using the JGI ‘bacterial genomic DNA isolation using CTAB’ protocol [37 ]. Short stretches of each CRISPR locus, encompassing at least a part of the leader sequence and the first spacer-repeat unit, were PCR amplified (for primers see S7 Table), and resolved on 2% agarose gels. Gels were stained with SYBR Safe Nucleic Acid Stain (Invitrogen) and nucleic acids were visualized using a G:BOX Chemi imager. A comparable method has previously been demonstrated to detect CRISPR adaptation if at least 0.4% of the culture obtained new spacers [32 (link)].