The first or second healthy leaves from the top were used for the DNA preparation. DNA sample was extracted from 3 mm2 of the small cut leaves. After the leaf samples were disrupted by ShakeMaster and zirconia beads (Hirata Corporation), genomic DNA was extracted from leaf chips of the transgenic cassava plants according to the protocol of Wizard Magnetic 96 DNA Plant System (Promega) using BIOMEK (Beckman Coulter) for the confirmation of gfp and hpt genes in genomic DNA of transgenic cassava. The genomic DNA was stored at -80°C until further use.
Wizard magnetic 96 dna plant system
Manufactured by Promega
Sourced in United States
The Wizard Magnetic 96 DNA Plant System is a laboratory equipment designed for the rapid and efficient extraction of DNA from plant samples. It utilizes magnetic bead technology to capture and purify DNA, providing a convenient and consistent method for high-throughput DNA isolation.
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5 protocols using wizard magnetic 96 dna plant system
1
Transgenic Cassava DNA Extraction
2
Genomic DNA Extraction and Transgene Detection in Cassava
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The rst or second youngest, healthy leaves of in vitro plants were used for genomic DNA preparation. Leaf samples were disrupted using a ShakeMaster and zirconia beads (Hirata Corporation), and genomic DNA extracted according to the protocol of Wizard Magnetic 96 DNA Plant System (Promega) using BIOMEK (Beckman Coulter). Genomic DNA was stored at -80ºC until further use.
The presence of gfp and hpt transgenes in transgenic cassava lines was con rmed by PCR analysis using KOD-FX Neo (Toyobo) and gene-speci c primers. The thermocylcer parameters used for ampli cation of gfp and hpt were 95°C for 5 min; 35 cycles of 98°C for 10 sec, 68°C for 30 sec, and 68°C for 30 sec; and a nal 5-min extension at 68°C. PCR products were analyzed by gel electrophoresis on a 1% (w/v) agarose gel stained with ethidium bromide. The primers used for PCR were as follows: GFP, 5'-CACTGGAGTTGTCCCAATTCTTGTTG-3' (forward) and 5'-CATGCCATGTGTAATCCCAGCA-3' (reverse); HPT, 5'-TCACCGCGACGTCTGTCGAG-3' (forward) and 5'-GCTCCATACAAGCCAACCAC-3' (reverse).
Total RNA extraction from FEC samples RNA was extracted from homogenous FEC tissues of KU50 and 60444. Total RNA was extracted from at least 100 mg FW (fresh weight) per biological replicate as described by Utsumi et al. (2017) and then stored at - 80 °С until use.
The presence of gfp and hpt transgenes in transgenic cassava lines was con rmed by PCR analysis using KOD-FX Neo (Toyobo) and gene-speci c primers. The thermocylcer parameters used for ampli cation of gfp and hpt were 95°C for 5 min; 35 cycles of 98°C for 10 sec, 68°C for 30 sec, and 68°C for 30 sec; and a nal 5-min extension at 68°C. PCR products were analyzed by gel electrophoresis on a 1% (w/v) agarose gel stained with ethidium bromide. The primers used for PCR were as follows: GFP, 5'-CACTGGAGTTGTCCCAATTCTTGTTG-3' (forward) and 5'-CATGCCATGTGTAATCCCAGCA-3' (reverse); HPT, 5'-TCACCGCGACGTCTGTCGAG-3' (forward) and 5'-GCTCCATACAAGCCAACCAC-3' (reverse).
Total RNA extraction from FEC samples RNA was extracted from homogenous FEC tissues of KU50 and 60444. Total RNA was extracted from at least 100 mg FW (fresh weight) per biological replicate as described by Utsumi et al. (2017) and then stored at - 80 °С until use.
3
Genomic DNA Extraction and Transgene Detection in Cassava
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The rst or second youngest, healthy leaves of in vitro plants were used for genomic DNA preparation. Leaf samples were disrupted using a ShakeMaster and zirconia beads (Hirata Corporation), and genomic DNA extracted according to the protocol of Wizard Magnetic 96 DNA Plant System (Promega) using BIOMEK (Beckman Coulter). Genomic DNA was stored at -80ºC until further use.
The presence of gfp and hpt transgenes in transgenic cassava lines was con rmed by PCR analysis using KOD-FX Neo (Toyobo) and gene-speci c primers. The thermocylcer parameters used for ampli cation of gfp and hpt were 95°C for 5 min; 35 cycles of 98°C for 10 sec, 68°C for 30 sec, and 68°C for 30 sec; and a nal 5-min extension at 68°C. PCR products were analyzed by gel electrophoresis on a 1% (w/v) agarose gel stained with ethidium bromide. The primers used for PCR were as follows: GFP, 5'-CACTGGAGTTGTCCCAATTCTTGTTG-3' (forward) and 5'-CATGCCATGTGTAATCCCAGCA-3' (reverse); HPT, 5'-TCACCGCGACGTCTGTCGAG-3' (forward) and 5'-GCTCCATACAAGCCAACCAC-3' (reverse).
Total RNA extraction from FEC samples RNA was extracted from homogenous FEC tissues of KU50 and 60444. Total RNA was extracted from at least 100 mg FW (fresh weight) per biological replicate as described by Utsumi et al. (2017) and then stored at - 80 °С until use.
The presence of gfp and hpt transgenes in transgenic cassava lines was con rmed by PCR analysis using KOD-FX Neo (Toyobo) and gene-speci c primers. The thermocylcer parameters used for ampli cation of gfp and hpt were 95°C for 5 min; 35 cycles of 98°C for 10 sec, 68°C for 30 sec, and 68°C for 30 sec; and a nal 5-min extension at 68°C. PCR products were analyzed by gel electrophoresis on a 1% (w/v) agarose gel stained with ethidium bromide. The primers used for PCR were as follows: GFP, 5'-CACTGGAGTTGTCCCAATTCTTGTTG-3' (forward) and 5'-CATGCCATGTGTAATCCCAGCA-3' (reverse); HPT, 5'-TCACCGCGACGTCTGTCGAG-3' (forward) and 5'-GCTCCATACAAGCCAACCAC-3' (reverse).
Total RNA extraction from FEC samples RNA was extracted from homogenous FEC tissues of KU50 and 60444. Total RNA was extracted from at least 100 mg FW (fresh weight) per biological replicate as described by Utsumi et al. (2017) and then stored at - 80 °С until use.
4
Wheat Cultivar Crossing and RIL Development
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Two bread wheat cultivars, ‘Victo’ (pedigree: NK-79-W-810/W-0010-E//W-1051-A) and ‘Spada’ (pedigree: TREBBO/KANSAS//(TR.TG) PSEUDOCERVINUM), were crossed, and 128 F7–8 RILs were developed by single-seed descent. For DNA extraction, one leaf of each line was ground using the Retsch MM300 Mixer Mill instrument (Newtown, PA, USA), then the Wizard Magnetic 96 DNA Plant System (Promega Italia, Milano, Italy) kit was used for DNA extraction and purification following the manufacturer’s instructions. All the germplasm was developed and maintained by CREA-Research Centre for Genomics and Bioinformatics in Fiorenzuola d’Arda (Piacenza, Italy).
5
Quantitative Detection of Bacterial Infection
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For bacterial infection estimation, leaves were collected from each individual tree every 3 months. Detection was performed as in [48 (link)]. Briefly, three to four mature leaves from recent flushes were randomly collected from the middle tree of each replicated plot every 3 months for 1 year. The petioles and midribs of leaves were excised, minced with a razor blade, lyophilized in a FreeZone 6 freeze–dry system (Labconco, Kansas City, MO, USA), and pulverized using a mini bead beater (Biospec products, Inc., Bartlesville, OK, USA). According to the manufacturer’s instructions, DNA from 100 mg of pulverized leaves was extracted using the Wizard Magnetic 96 DNA Plant System (Promega Corporation, Madison, WI, USA). DNA was quantified using a microplate reader (Synergy HTX Multimode Reader, Biotek Instruments, Inc., Winooski, VT, USA) and normalized to 10 ng/µL. CLas was detected by quantitative real-time polymerase chain reaction (qRT-PCR) using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers and probes used for detection were HLBas and HLBr and probe HLBp, respectively, as described in [49 (link)]. Samples with a Ct value of less than 36 were considered CLas-positive.
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