In order to provide enough donor fragments for HDR, both vectors and the free 549 bp left homology armed-DRT and the 670 bp armed-DRT with two homologous arms were co-introduced into rice (japonica cv. Zhonghua 11) calli with a molar ratio of 1:20 by particle bombardment Two days after bombardment, DNA from the calli was extracted using a DNA Quick Plant System (Tiangen, Beijing, China). PCR amplification was performed using EASY Taq polymerase (TransGen Biotech) employing 200 ng of genomic DNA as template. Each callus was tested individually by PCR and sequencing. The PCR products were generated using the allele-specific primer set ALSTestF/T2MR (Supplementary Table S1 ) with the upstream primer located in the genome sequence of the ALS gene outside of the left homology arm, whereas the downstream primer was an allele-specific primer (Fig. 1A ). The obtained amplicons were cloned into the cloning vector pEasy-Blunt Zero (TransGen Biotech). At least 10 positive colonies for each sample were sequenced.
Peasy blunt zero
Manufactured by Transgene
Sourced in China
PEASY-Blunt Zero is a versatile lab equipment product designed for precision DNA cloning. It enables the efficient blunt-end ligation of DNA fragments, facilitating the construction of recombinant plasmids. The core function of PEASY-Blunt Zero is to provide a reliable and streamlined solution for DNA cloning procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dna quick plant system, by Tiangen Biotech (1 mentions)
Easytaq polymerase, by Transgene (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Smarter race 5 3 kit, by Takara Bio (1 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Transzol up rna extraction kit, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for pcr, by Transgene (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
7 protocols using peasy blunt zero
1
HDR Donor Fragment Generation in Rice
2
Validating Reference Genes for qRT-PCR
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The fourteen common RGs were amplified according to the references or based on primers designed by Primer-BLAST of the RNA sequence data of leaves and fruits (Table 1 ). Specific primers for the candidate RGs from our RNA-sequencing data were designed using Primer 3 (http://primer3.ut.ee/ ) (Table 2 ). All PCR amplicon lengths were between 80–200 bp. All primers were synthesized by a commercial supplier (Biosune, Hangzhou, China).
To check the specificity of all primers, the cDNA of each sample was amplified by PCR, and the amplified products were separated by electrophoresis on 3% agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and cloned into pEASY-Blunt zero (Transgen, Beijing, China) followed by sequencing.
The quantification cycle (Cq) values obtained by qRT-PCR on a standard curve generated from a fourfold dilution series of one sample at six dilution points for three technical replicates were used to draw the standard curve to get R2 and slope values. The PCR amplification efficiency of each primer was calculated using the equation: E(Efficiency)% = (10 [−1∕slope] −1) ×100%.
To check the specificity of all primers, the cDNA of each sample was amplified by PCR, and the amplified products were separated by electrophoresis on 3% agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and cloned into pEASY-Blunt zero (Transgen, Beijing, China) followed by sequencing.
The quantification cycle (Cq) values obtained by qRT-PCR on a standard curve generated from a fourfold dilution series of one sample at six dilution points for three technical replicates were used to draw the standard curve to get R2 and slope values. The PCR amplification efficiency of each primer was calculated using the equation: E(Efficiency)% = (10 [−1∕slope] −1) ×100%.
3
Amplification and Sequencing of PAstV Genome
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The full-length genome of PAstV isolate was divided into six fragments (Table S2 in Supplementary Data S1 ), which were amplified by RT-PCR and cloned into the vector pEASY Blunt Zero (TransGen, Beijing, China) in accordance with the manufacturer’s protocol. The 5′- and 3′-end sequences were obtained using SMARTer RACE 5′/3′ Kit) (Takara®, Beijing, China). The PCR products were sent to BioSune Biotechnology Co., Ltd. (Shanghai, China) for sequencing. All of the sequencing reactions were done in quintuplicate, and all sequences were confirmed by sequencing both strands.
Sequence analysis was conducted using the EditSeq tool included with the Lasergene DNASTARTM 7.0 software package (DNASTAR Inc., Madison, WI, USA).
Sequence analysis was conducted using the EditSeq tool included with the Lasergene DNASTARTM 7.0 software package (DNASTAR Inc., Madison, WI, USA).
4
Cloning and Sequencing Splice Variants
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Total RNAs from fifth-instar P. rapae larvae were extracted using the TRIzol (Invitrogen, Carlsbad, CA, US) according to the manufacturer’s instructions. Single-strand complementary DNA (cDNA), synthesized from 1000ng of total RNA using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China), were as then used as templates for PCR reactions. The PCR primers for investigating the splice variants were designed with primer5 (Table S1 ) [18 (link)]. PCR products were separated by electrophoresis on 1% agarose gel. The gel where target fragments located were cut off and DNA were purified with Agarose Gel DNA Extraction Kit-250 prep (Esay-Do, Hangzhou, China). Then the purified PCR products were cloned into pEASY-Blunt Zero (Transgen, Beijing, China) and transformed into DH5α competent E. coli cells (Vazyme, Nanjing, China). Positive clones were sequenced by second-generation sequencing (Sunya, Hangzhou, China).
5
Yeast Assay for Lead Resistance
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The coding sequences of FtMTPC2, FtZFP14, FtPCSL, FtVCE1a, FtNramp3, and FtPCS1 from the cDNA library constructed by RNA-Seq were amplified, and the fragments were subcloned into pEASY-Blunt Zero (TransGen Biotech, China) and sequenced. The correct coding DNA sequence (CDS) fragments were cloned into a pYES2 vector using the ClonExpress II One Step Cloning kit (Vazyme, China) for expression in yeast. Cells of the Pb- and cadmium-sensitive yeast strain Δycf1 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 YCF1::kanMX4) carrying the empty vector (pYES2), pYES2-FtMTPC2, pYES2-FtZFP14, pYES2-FtPCSL, pYES2-FtVCE1a, pYES2-FtNramp3, or pYES2-FtPCS were grown in medium at 30 °C. To analyse Pb resistance, the yeast cells were cultured in SD-Ura (2% glucose) medium at 30 °C until reaching an OD600 of 2.0 and then serially diluted (10− 1, 10− 2, 10− 3, 10− 4) with 1× TE buffer. The cells were spotted onto an SD-Ura (2% galactose) medium plate with Pb2+ (0, 40, or 80 μmol/L) and cultured at 30 °C in an incubator for 4 d. Yeast cells were inoculated onto SD-Ura (2% galactose) medium with 0 or 40 μmol/L Pb2+ at a 1:1000 ratio to determine the growth curve.
6
Cloning and Sequencing of ApCtf1β Genes
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According to the manufacturer’s instructions, the total RNA of A. phaeospermum was extracted using the TransZol Up RNA Extraction Kit (TransGen). The extracted products were tested for purity and integrity via agarose electrophoresis and microspectrophotometry. According to the manufacturer’s instructions, RNA that met the target requirements was reverse-transcribed to synthesize cDNA for backup using the All-in-One First-Strand cDNA Synthesis Super Mix for PCR (TransGen a). The homologous primers ApCtf1β-F/ApCtf1β-R (Supplementary Table S1 ) were designed based on the ApCtf1β1 and ApCtf1β2 genes (MK789640 and MK789641) in the NCBI database. PCR amplification was performed using A. phaeospermum cDNA as the template. The PCR products were detected via 1% agarose gel electrophoresis, recovered, and ligated to the cloning vector pEASY-Blunt Zero (TransGen) to obtain the recombinant vector. The ligated products were transformed into receptor cells DH5α. Positive transformants were picked following verification via colony PCR and sent to Tsingke for sequencing to confirm that the target genes were not mutated.
7
CRISPR/Cas9 Editing of Cucumber TEN Gene
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To generate CRISPR/Cas9 engineered mutations in the TEN gene, a binary CRIPSR/Cas9 vector pBSE402 plus a 35S-GFP expression cassette was modified from pBSE401a 54 . For assembly of TEN sgRNA into pBSE402, equal volumes of 100 μM forward and reverse primers were mixed, incubated at 95°C for 5 min, and slowly cooled to room temperature, resulting in a double stranded DNA fragment with sticky BsaI ends. This short DNA fragment was then assembled into pBSE402, by restriction fragment ligation, using BsaI and T4 Ligase (New England Biolabs). Primers are shown in Supplementary Table 8. Agrobacterium tumefacines strain EH105, carrying a pBSE402-TEN construct, was used to transform the cucumber inbred line CU2, using cotyledonary nodes as explants, as previously described 55 . Shoot regeneration, elongation and rooting processes strictly followed normative procedures.
Genomic DNA was extracted from the positive transgenic plants using the DNeasy Plant Mini Kit (Qiagen, Cat. #69104). PCR was performed using gene-specific primers (Supplementary Table 8). PCR products were cloned into pEASY-Blunt Zero (TRANSGEN BIOTECH, Cat. #CB501) and the various alleles for the TEN gene were identified by sequencing.
Genomic DNA was extracted from the positive transgenic plants using the DNeasy Plant Mini Kit (Qiagen, Cat. #69104). PCR was performed using gene-specific primers (Supplementary Table 8). PCR products were cloned into pEASY-Blunt Zero (TRANSGEN BIOTECH, Cat. #CB501) and the various alleles for the TEN gene were identified by sequencing.
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