DNA purity was estimated using a spectrophotometer, (Nanodrop 2000; Thermo Fisher Scientific). DNA yield was estimated, using a fluorimeter and fluorescent DNA-binding dye (QubitTM dsDNA BR Assay Kit; Thermo Fisher Scientific), according to the manufacturer´s instructions. DNA integrity was checked by agarose gel electrophoresis.
To test the effectiveness of the reported DNA extraction protocol, we performed a series of small-scale parallel DNA extractions, with and without the sorbitol pre-wash step. Leaves of several plant genera regarded as “demanding” in our laboratory were tested, including A. occidentale (Anacardiaceae) (Cashew), E. grandis (Myrtaceae), Pereskia aculeata (Cactaceae), several different species of the genera Diplusodon and Lafoensia (both Lythraceae). DNA yields and purity were estimated spectrophotometrically. DNA quality was also inferred by a simple PCR amplification assay using the nuclear ribosomal ITS marker. Each reaction contained 1 X PCR buffer with 2.0 mM MgSO4, 0.2 mM dNTP’s, 0.2 M Trehalose, 0.3 μM each of the universal primers An5 and An4 [21 (link)], 1 U Taq DNA polymerase and 1 μl undiluted DNA. PCR cycling consisted of two minutes initial denaturation at 95°C then 35 cycles of 20 seconds at 95°C, 40 seconds at 55°C and 80 seconds at 72°C, followed by 7 minutes at 72°C. PCR products were analyzed using an ethidium bromide stained 1.5% w/v agarose gel, where the expected band size of the ITS fragment was approximately 650 bp.
To test the effectiveness of the reported DNA extraction protocol, we performed a series of small-scale parallel DNA extractions, with and without the sorbitol pre-wash step. Leaves of several plant genera regarded as “demanding” in our laboratory were tested, including A. occidentale (Anacardiaceae) (Cashew), E. grandis (Myrtaceae), Pereskia aculeata (Cactaceae), several different species of the genera Diplusodon and Lafoensia (both Lythraceae). DNA yields and purity were estimated spectrophotometrically. DNA quality was also inferred by a simple PCR amplification assay using the nuclear ribosomal ITS marker. Each reaction contained 1 X PCR buffer with 2.0 mM MgSO4, 0.2 mM dNTP’s, 0.2 M Trehalose, 0.3 μM each of the universal primers An5 and An4 [21 (link)], 1 U Taq DNA polymerase and 1 μl undiluted DNA. PCR cycling consisted of two minutes initial denaturation at 95°C then 35 cycles of 20 seconds at 95°C, 40 seconds at 55°C and 80 seconds at 72°C, followed by 7 minutes at 72°C. PCR products were analyzed using an ethidium bromide stained 1.5% w/v agarose gel, where the expected band size of the ITS fragment was approximately 650 bp.
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