At T0, leaf tissue was collected from rooted putative transgenic plants before transplanting to soil to screen for low copy, simple events. At T1, 1 cm leaf punches were collected at the V4 stage for zygosity analysis. DNA was extracted with a Qiagen MagAttract 96 DNA Plant Core Kit (Qiagen, Gaithersburg, MD) using KingFisher magnetic particle processors (Thermo Scientific, Waltham, MA) following manufacture’s recommendation.
Magattract 96 dna plant core kit
Manufactured by Qiagen
Sourced in United States
The MagAttract 96 DNA Plant Core Kit is a laboratory equipment product designed for the rapid and efficient extraction of high-quality DNA from plant samples. It utilizes magnetic bead-based technology to streamline the DNA purification process, enabling consistent and reliable results.
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Lab products found in correlation
6 protocols using magattract 96 dna plant core kit
1
Screening Transgenic Leaf Samples
2
Genomic DNA Extraction from Fungal Mycelia
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The total genomic DNA was extracted from lyophilized mycelia obtained from 10-day-old cultures grown on PDA as described by Ghimire et al. [67 ] using Qiagen MagAttract96 DNA Plant Core Kit according to the manufacturer’s instructions. The quality and concentration of extracted DNA were estimated using the NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Walthum, MA) and visualized in 1% agarose gel stained with SYBR Safe DNA gel stain under ultra violet-light (UVP BioImaging Systems, Upland, CA). DNA was stored at -20°C until further use.
3
DNA Extraction from Plant Tissues
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DNA was extracted from leaves and seeds with the Nucleospin 96 plant kit (Macherey-Nagel, Bethlehem, PA) following the protocols described in Isabel et al. (2013 ). DNA from seedlings was extracted with MagAttract 96 DNA Plant Core Kit (Qiagen, Mississauga, ON) according to the manufacturer's instructions.
4
Maize Genomic DNA Extraction
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DNA samples from the inbred lines B73, Mo17 and the nested association mapping (NAM) founders (19 (link)) were extracted from 6-day-old seedling tissue using the DNeasy Plant Maxi Kit [QIAGEN (Valencia, CA, USA), No. 68163]. The 232 B73xMo17 recombinant inbred lines (IBM RILs) (20 (link)) and the 192 F2 individuals were extracted from 6-day-old seedling leaf tissue using the MagAttract 96 DNA Plant Core Kit [QIAGEN (Valencia, CA, USA), No. 67163]. Samples were normalized using the Qubit dsDNA Broad Range Assay [ThermoFisher (Waltham, MA, USA), no Q32853].
5
Molecular Characterization of Epichloë Endophytes
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Infection status for each individual plant (two to three tillers per plant) was initially determined by an immunoblot assay that utilizes monoclonal antibodies to detect Epichloë endophytes (Phytoscreen Immunoblot Kit; Agrinostics, Watkinsville, Georgia). The infection status was reconfirmed using a PCR-based method that could also detect endophyte genotypic variation. DNA was isolated from freeze-dried tillers with the MagAttract 96 DNA Plant Core Kit (Qiagen Inc., Germantown, Maryland) according to manufacturer's instructions. Five multiplex primer combinations (SUPPLEMENTARY TABLE 2) were used for PCR to genetically characterize the endophyte with respect to infection status (tefA), mating type (MTAand MTB), and presence of peramine (PER), ergot (EAS), loline (LOL), indole-diterpene (IDT/LTM) alkaloid genes, as described in Charlton et al. (2014) . The endophytes were grouped on the basis of presence or absence of these PCR markers, and the alkaloid potential of the endophyte was predicted.
6
Illumina-based Microbiome Profiling of Root Samples
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Prior to DNA extraction, we cut each root sample into approximately 2 cm pieces and collected them randomly into sterilized tubes. The genomic DNA of each root sample was extracted using a Qiagen MagAttract 96 DNA Plant Core Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's instructions.
Pair-end library preparation for MiSeq sequencing was conducted using the twostep tailed PCR method described on Illumina (Illumina, San Diego, CA, USA). We The 3' end to 18 bases and 19 bases of each primer (forward and reverse, respectively)
were 16S rRNA universal sequences (Chelius and Triplett 2001) . The 19-30 base from the 3' end of the forward primer was the unique molecular identifier (Fields et al., 2020) .
The other regions were the Illumina overhang adapter sequences. The second-round PCR was performed using primer pairs with 16 unique indices: D501-D508 and A501-A508 (forward) and D701-D712 and A701-A712 (reverse) (Illumina).
The DNA concentration of the purified PCR products was adjusted and pooled into two different tubes, as the MiSeq run was performed in two separate runs. The samples used for the experiments are listed in Table S1. The paired-end libraries were mixed with 3% PhiX DNA spike-in control and used for sequencing on the MiSeq platform using Illumina MiSeq v.3 Reagent kit for 2 x 300 bp PE.
Pair-end library preparation for MiSeq sequencing was conducted using the twostep tailed PCR method described on Illumina (Illumina, San Diego, CA, USA). We The 3' end to 18 bases and 19 bases of each primer (forward and reverse, respectively)
were 16S rRNA universal sequences (Chelius and Triplett 2001) . The 19-30 base from the 3' end of the forward primer was the unique molecular identifier (Fields et al., 2020) .
The other regions were the Illumina overhang adapter sequences. The second-round PCR was performed using primer pairs with 16 unique indices: D501-D508 and A501-A508 (forward) and D701-D712 and A701-A712 (reverse) (Illumina).
The DNA concentration of the purified PCR products was adjusted and pooled into two different tubes, as the MiSeq run was performed in two separate runs. The samples used for the experiments are listed in Table S1. The paired-end libraries were mixed with 3% PhiX DNA spike-in control and used for sequencing on the MiSeq platform using Illumina MiSeq v.3 Reagent kit for 2 x 300 bp PE.
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