rbcL DNA-Seq of Arbutus sp.: leaf
Approximately 100 mg of wet leaf tissue were flash frozen with liquid nitrogen and homogenized using a manual mortar and pestle or Bel-Art SP Scienceware Disposable Polypropylene Pestles (catalog number: F199230001, Bel-Art, Wayne, New Jersey, USA) attached to a DEWALT Bare-Tool DCD760B 1/2-Inch 18-Volt Cordless Compact Drill/Driver (catalog number: DCD760, Dewalt, Towson, Maryland, USA) in 400 l of Buffer AP1 and 4 l RNase A stock solution from the DNeasy Plant Mini Kit (catalog number: 69104, QIAGEN, Hilden, Germany) for low DNA yield samples. DNA was extracted using the DNeasy Plant Mini Kit (catalog number: 69104, QIAGEN, Hilden, Germany). Barcoding region of rbcL was amplified in an Axygen Maxygene II Thermal Cycler (catalog number: THERM-1001, Axygen a Corning brand, New York, USA) using GoTaq Green Master Mix (catalog number: M7122, Promega, Madison, Wisconsin, USA). Primers were generated using Primer3: rbcL-F 5-TCG TTA CAA AGG GCG ATG CT-3', rbcL-R 5-TGT CCT AAA GTT CCG CCA CC-3'. The Sanger sequencing was performed by Eton Bioscience Inc. and Genewiz from Azenta Life Sciences using ABI-3730 xl DNA analyzer.