The cotton genomic DNA was extracted from leaves by Plant Genome Extraction Kit (TianGen, Beijing, China) according to the instruction manual. To determine the T-DNA inserted copy numbers of GhBOP1 transgenic plants, a southern blot analysis was performed according to a previously described method59 (link). Briefly, a total of 20 μg genomic DNA was digested by Hind III for 24 h, then separated by electrophoresis on a 0.8% agarose gel. After that, transfer the separated DNA to the nylon membrane (Hybond-N+; Amersham, Buckinghamshire, UK). The amplicon of NPTII fragment was used as a probe, which was labeled with α-[32P] through a random primer labeling kit (Promega, Madison, WI, United States).
Plant genome extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The Plant Genome Extraction Kit is a laboratory equipment designed for the extraction and purification of genomic DNA from plant samples. It provides a straightforward and efficient method to obtain high-quality plant DNA for various downstream applications, such as PCR, sequencing, and genetic analysis.
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Lab products found in correlation
8 protocols using plant genome extraction kit
1
Determining T-DNA Copy Number in Transgenic Cotton
2
Genomic DNA Extraction of Lilium Species
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Fresh leaves of nine Lilium species were sampled (Table 5 ). Specimens were deposited in the herbarium of the Institute of Botany, Chinese Academy of Sciences (PE) (Table 5 ). Total genomic DNA was extracted using a plant genome extraction kit (Tiangen, Beijing, China). Subsequently, DNA concentration was measured using a NanoDrop spectrophotometer 2000 (Thermo Fisher Scientific, America).
Sampled species and their voucher specimens used in this study.
| Species | Section | Voucher | Locality |
|---|---|---|---|
| L. fargesii | Lophophorum | BOP040593 | Shaanxi |
| L. brownii | Leucolirion 6b | BOP040602 | Hubei |
| L. lancifolium | Sinomartagon | BOP040607 | Hubei |
| L. nepalense var.ochraceum | Lilium–Nomocharis | BOP040618 | Yunnan |
| L. leucanthum | Leucolirion 6a | BOP040622 | Chongqing |
| L. davidii var. willmottiae | Sinomartagon | BOP040624 | Yunan |
| L. duchartrei | Sinomartagon | BOP040925 | Sichuan |
| L. bakerianum | Lophophorum | BOP040929 | Yunnan |
| L. henryi | Leucolirion 6a | BOP040933 | Hubei |
3
Rapid DNA Extraction and Amplification
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Cas12a(LbCpf1) Nuclease, RNase Inhibitor and Ultra-Rapid Nucleic Acid Release Agents (for DNA) were purchased from Tolo (Shanghai, China), BBI (Shanghai, China) and Weifang Ample Future Biotechnology (Weifang, China), respectively. The plant genome extraction kit was purchased from Tiangen biochemical Technology (Beijing, China). Agarose, Axygen plasmid DNA small quantity kit, and Axygen DNA gel recovery kit were purchased from Beijing Bingda Biotechnology（Beijing, China）. The RPA kit was purchased from TwistDx Ltd. (Cambridge, MA, USA). The synthesis of oligonucleotides and standard plasmid molecules, and the sequencing of amplified products were completed by Sangon Biotech (Shanghai, China).
GM corn, alfalfa, rapeseed, soybeans, and sugar beets were purchased from Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) and American Oil Chemists Society (AOCS, Urbana, IL, USA). Transgenic cotton and non-GM corn and soybeans were provided by the crop Ecology and Environmental Safety Monitoring Center of the Ministry of Agriculture of the people's Republic of China. The materials were listed inTable S1 .
GM corn, alfalfa, rapeseed, soybeans, and sugar beets were purchased from Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) and American Oil Chemists Society (AOCS, Urbana, IL, USA). Transgenic cotton and non-GM corn and soybeans were provided by the crop Ecology and Environmental Safety Monitoring Center of the Ministry of Agriculture of the people's Republic of China. The materials were listed in
4
Extraction and Validation of Transgenic Cotton
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DNA was extracted from T0 transgenic cotton plants and wild type as control using a Plant Genome Extraction Kit (TIANGEN BIOTECH, Beijing, China). Specific primers for AacCas12b and sgRNA sequences were used to check confirm plant transformation. The targeted sites were amplified using site‐specific primers, and the amplicons were ligated in pGEMT‐Easy vector using T4 DNA ligase (Promega, Madison, USA). The vector was transformed into an E. coli strain using Top10, and positive clones were used for DNA Sanger sequencing.
5
Establishing Transgenic 84K Poplar Lines
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The constructed 35S::PagSTOMAGEN-GFP was introduced into the Agrobacterium strain GV3101 using the freeze–thaw method [71 (link)]. The Agrobacterium tumefaciens-mediated transformation of 84K poplar was performed as described previously [35 (link)]. The infected leaves were transplanted to MS agar medium containing 30 mg∙L−1 kanamycin and 200 mg∙L−1 timentin for screening. Regenerating buds were transferred to 1/2 MS agar medium with 30 mg∙L−1 kanamycin and 200 mg∙L−1 timentin until rooting.
Genomic DNA was extracted from each presumptive transgenic line and the WT plant using a Plant Genome Extraction Kit (Tiangen, China, Cat DP320). Transformation was verified using primers the 35S-F and PagSTOMAGEN-R (Table S1 ). The expression level of PagSTOMAGEN was detected using RT-qPCR. The 84K transgenic lines identified via PCR and RT-qPCR were used for propagation. One-month-old transgenic plants were transplanted to the greenhouse (16 h light/8 h dark, 22–25 °C, relative humidity: 40–45%).
Genomic DNA was extracted from each presumptive transgenic line and the WT plant using a Plant Genome Extraction Kit (Tiangen, China, Cat DP320). Transformation was verified using primers the 35S-F and PagSTOMAGEN-R (
6
Transgenic Cotton Genome Editing Analysis
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Genomic DNA of the cotton plants was extracted with Plant Genome Extraction Kit (TIANGEN, Beijing, China). For transgenic positivity check, specific primers of Cas9 sequence were used in PCR analysis. Southern blotting was conducted with NPT II fragment as the DNA probe to detect DsRed2 copies using DIG High Prime DNA Labelling and Detection Starter Kit II (Roche, Basel, Switzerland). Partial GhCLA1 sequences that covered sgRNA7‐sgRNA8 sites and sgRNA9‐sgRNA10 sites, respectively, were used for mutation genotyping in independent transgenic lines. The obtained PCR products were ligated in pGEMT‐Easy vector for TA cloning with T4 DNA ligase (Promega, Madison, WI). After reaction, the ligated products were transformed into Escherichia coli strain Top10, and positive clones were applied for DNA Sanger sequencing.
7
Genomic DNA Extraction and Mutation Analysis
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Genomic DNA of the cotton plants were extracted with Plant Genome Extraction Kit (TIANGEN, Beijing, China). For transgenic positivity check, specific primers of Cas9 sequence were used in PCR analysis. Partial GhCLA1 sequences that covered transgenic sgRNA1-sgRNA2 sites, sgRNA3-sgRNA4 sites, sgRNA5-sgRNA6 sites, and their predicted off-target sites, respectively, were used for mutation genotyping in independent lines. The obtained PCR products were ligated in pGEMT-Easy vector for TA cloning with T4 DNA ligase (Promega, Madison, WI, USA). After reaction, the ligated products were transformed into E. coli strain Top10 and positive clones were applied for DNA Sanger sequencing.
8
Cotton Genomic DNA Extraction
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The cotton genomic DNA was isolated and purified from calli by Plant Genome Extraction Kit (TianGen, Beijing, China) according to the instruction manual. The DNA of samples was diluted into 20 ng•μL -1 for PCR reaction. The PCR primers were listed in Table 2.
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