Extract n amp plant pcr kit

DNA was extracted using Extract‐N‐Amp™ Plant PCR Kits (Sigma‐Aldrich, Oakville, ON, Canada). All samples were genotyped for 18 variable microsatellite loci using PCR and genotyping protocols as previously described by Graignic et al. (2013). Five different multiplex PCR sets were used, and 37 cycles in the PCR amplification were processed (see Table S1).

Total genomic DNA was extracted from silica-dried leaves using Extract-N-Amp™ Plant PCR Kits (SIGMA - Aldrich, St. Louis, MO, USA) abiding by the supplier’s instructions. Fifteen nuclear SSR markers previously selected for studying the genetic diversity of Prunus species in the Mediterranean region were used for this study: UDP96–001, UDP96–018, UDP96–003, UDP97–401, UDP98–408, UDP98–409, pchgms1, pchgms3, BPPCT017, BPPCT001, BPPCT007, BPPCT025, BPPCT036, CPSCT018, CPDCT045 [34 (link)–38 (link)]. We amplified these 15 SSR markers into three multiplexed PCRs, using one of the FAM, HEX or NED fluorophore-labeled primers (PE Applied Biosystems, Warrington, UK). Multiplexed PCRs were carried out with the Extract-N-Amp PCR Ready Mix (SIGMA - Aldrich, St. Louis, MO, USA) in a final volume of 10 μl, containing 5 μl of a SIGMA Master Mix 2X, 0.4 μl of primer mix at 5 μM, 1.6 μl of ultrapure water and 1 μl of template dNTPs. 3 μl of PCR product was mixed with 15 μl of formamide and 0.2 μl of Genescan™ 500 LIZ size standard (Applied Biosystems, Foster City, USA), and GeneScan was performed with the ABI PRISM 3130 XL 16 capillary-sequencer (Applied Biosystems, Foster City, USA).

DNA extraction and PCR amplification of the variable region 4 (V4) of the 16S rRNA gene using Illumina adapted universal primers 515F/806R39 was conducted using the direct PCR protocol [Extract-N-Amp Plant PCR kit (Sigma-Aldrich, Inc.)] as previously described [24 (link),25 (link),26 (link)]. Briefly, PCRs were conducted in triplicate in 96 wells plate [denaturation for 3 min at 94 °C; 35 cycles (98 °C, 60 s; 55 °C, 60 s; 72 °C, 60 s) followed by elongation for 10 min at 72 °C]. Positive amplicons were pooled in equimolar concentrations into a composite sample that was size selected (300–500 bp) using agarose gel to reduce non-specific products from host DNA. Sequencing was performed on the Illumina MiSeq platform with the addition of 20% PhiX, and generating paired-end reads of 175b in length in each direction.

The PCR analysis of the regenerated plants was conducted using the Extract-N-Amp Plant PCR kit (Sigma-Aldrich, St. Louis, MO, USA). A pair of PCR primers located within the ProINS-Tf expression cassette were used for PCR amplification screening of positive transgenic plants. The amplification reaction condition was as follows: 94 °C for 5 min, 35 cycles of 94 °C, 1 min; 60 °C 1 min; and 72 °C for 1 min followed by 72 °C for 10 min. The PCR products were resolved in 1% agarose gel by electrophoresis.

Fresh fecal samples were collected during the last week of the experiment for gut microbial characterization. Bacterial genomic DNA was extracted from frozen fecal samples stored at −80 °C. A smear approximately 5 mm² (roughly the size of a pencil eraser) was taken from a fecal sample using a sterile swab in a test tube. DNA extraction and PCR amplification of variable region 4 (V4) of the 16S rRNA gene using Illumina-adapted universal primers 515F/806R was conducted using the direct PCR protocol (Extract-N-Amp Plant PCR kit (Sigma-Aldrich, Inc., St. Louis, MO, USA)) as previously described [17 (link),18 (link)]. Briefly, PCRs were conducted in triplicate in 96-well plates (denaturation for 3 min at 94 °C; 35 cycles (98 °C, 60 s; 55 °C, 60 s; 72 °C, 60 s) followed by elongation for 10 min at 72 °C). Positive amplicons were pooled in equimolar concentrations into a composite sample that was size selected (300–500 bp) using agarose gel to reduce non-specific products from the host DNA. Sequencing was performed on the Illumina MiSeq platform (San Diego, CA, USA) with the addition of 20% PhiX (illumina cat #15017666, San Diego, CA, USA) and paired-end reads of 175 b in length were generated in each direction.

Genomic DNA was extracted from the dried basidiocarps of herbarium materials using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States), QIAamp DNA Micro Kit (Qiagen), and the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, United States), following protocols from the manufacturers, and was diluted as a template for subsequent amplification. PCR amplification targeted the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rRNA), the universal DNA barcode for identification of fungi (Schoch et al., 2012 (link)), and the nuclear large ribosomal subunit (nLSU) region. Amplification was carried out using the fungal-specific primer sets ITS1F/ITS4b (Gardes and Bruns, 1996 ) and ITS1/ITS4 (White et al., 1990 ) for the ITS region and LR0R and LR5 for the nLSU region (Vilgalys and Hester, 1990 (link); Rehner and Samuels, 1994 (link)). Purified PCR products were sequenced using DNA ABI 3730 XL automated sequencers (Applied Biosystems) by Macrogen Inc. (Seoul, Korea), by Eurofins Genomics (Ebersberg, Germany), and by the Beijing Genomics Institute (Beijing, China). All newly generated sequences of poroid and corticoid species from Uzbekistan were submitted to GenBank (Table 1).