The cetyltrimethylammonium bromide (CTAB) method was used to extract genomic DNA from the leaves of maize seedlings [28 (link)]. PCR amplifications of relevant regions in transgenic plants were accomplished using the specific primers in Table S1 . For genotyping plants in T0 and F1 generations, the PCR products of relevant regions were purified and introduced into the pEASY-T5 Zero Cloning vector (TransGen Biotech, Beijing, China) for DNA sequencing. The sequencing chromatograms were meticulously analyzed for exact patterns that might indicate the mutation types, i.e., homozygous, monoallelic, or diallelic mutations. The co-segregating molecular markers were designed according to mutations of target genes (Table S1 ), as described previously [29 (link)]. The PCR products were analyzed in F2 generation using polyacrylamide gel electrophoresis.
Peasy t5 zero cloning vector
Manufactured by Transgene
Sourced in China
The PEASY-T5 Zero cloning vector is a plasmid designed for high-efficiency cloning and expression of heterologous genes in Escherichia coli. The vector features a T5 promoter for strong, inducible expression of the target gene and lacks a multiple cloning site, allowing for direct insertion of the gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Axygen dna gel extraction kit, by Corning (1 mentions)
Kod fx neo, by Toyobo (1 mentions)
Microelute genomic dna kit, by Omega Bio-Tek (1 mentions)
E z n a, by Omega Bio-Tek (1 mentions)
Axyprep plasmid miniprep kit, by Corning (1 mentions)
Sanger sequencing, by Sangon (1 mentions)
Viafect, by Promega (1 mentions)
Transdirect animal tissue pcr kit, by Transgene (1 mentions)
Automated dna sequencer, by Thermo Fisher Scientific (1 mentions)
Diaspin dna gel extraction kit, by Sangon (1 mentions)
Trans5α chemically competent cell, by Transgene (1 mentions)
Haeiii, by Takara Bio (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Ni nta resin, by GE Healthcare (1 mentions)
5 full race kit, by Takara Bio (1 mentions)
Dh5α competent cells, by Takara Bio (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Plasmid mini kit, by Omega Bio-Tek (1 mentions)
22 protocols using peasy t5 zero cloning vector
1
Genotyping Transgenic Maize Plants
2
Comparative Analysis of Gene Cassettes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine whether each cassette gene PCR amplicon with the same size had the same content, cassette PCR products were digested with AluI or RsaI (New England Biolabs, Beijing, China). The PCR products with the same RFLP pattern were considered to contain same gene cassettes. To analyze the sequences of the gene cassettes, 1 or 2 cassette amplicons representative of each restriction profile were excised, purified and ligated into pEASY®-T5 Zero Cloning Vector (TransGen Biotech, Beijing, China). The ligation mixture was transformed into Trans-T1 Phage Resistant Chemically Competent Cell (TransGen Biotech, Beijing, China) and recombinants were selected on Luria agar containing ampicillin (100 mg/L). Recombinant plasmid DNA was purified by standard methods and subjected to sequencing at Nanjing GenScript Ltd. (Nanjing, China). The nucleotide sequences were analyzed and compared with available sequences in GenBank using the BLAST program by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/ ).
3
Genotyping Fruiting Body Patterns via SRAP Diversity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nine forward primers and 17 reverse primers for SRAPs were combined two by two for the diversity analysis of the set of fruiting body pattern genotypes (Table 2 ) [23 ,24 ]. Each amplification was carried out in a 20-μL reaction mixture under the following PCR conditions according to Li et al. [23 ].
Specific bands associated with the fruiting body pattern were screened, excised, and purified with an AXYGEN DNA Gel Extraction Kit (Axygen, Union City, CA). The extracted DNA was ligated into the pEASY®-T5 zero cloning vector (TransGen Biotech Co., Beijing, China), and the positive clones were screened for DNA sequencing. The DNA was sequenced by a commercial DNA sequencing service (Jinweizhi Biotechnology Co., Ltd., Suzhou, China). Data analysis and sequence alignments were conducted using DNAMAN® version 5.2.9 (Lynnon Bio Soft, San Ramon, CA) and the GenBank database (https://www.ncbi.nlm.nih.gov/ ).
Specific bands associated with the fruiting body pattern were screened, excised, and purified with an AXYGEN DNA Gel Extraction Kit (Axygen, Union City, CA). The extracted DNA was ligated into the pEASY®-T5 zero cloning vector (TransGen Biotech Co., Beijing, China), and the positive clones were screened for DNA sequencing. The DNA was sequenced by a commercial DNA sequencing service (Jinweizhi Biotechnology Co., Ltd., Suzhou, China). Data analysis and sequence alignments were conducted using DNAMAN® version 5.2.9 (Lynnon Bio Soft, San Ramon, CA) and the GenBank database (
4
Amplification and Analysis of Sweet Potato Ibβfruct2 Promoters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The promoters of Ibβfruct2 genes and their genomic sequences were amplified from genomic DNA of the eight sweet potato cultivars listed in section 2.1 by PCR using KOD FX Neo (TOYOBO) and the primers Pft2-2.5F and IbβfrTct2-mRNA-Rev (Table S1 http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ; Lescot et al., 2002 (link)).
5
Genomic DNA Bisulfite Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted using an E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-Tek) according to the manufacturer's protocol. About 500 ng to 1 μg of genomic DNA was bisulfate-treated followed with PCR according to published protocols (Kerjean et al., 2000 (link), Kim et al., 2007 (link)). The PCR products were cloned into pEASY-T5 Zero Cloning Vector (TransGen Biotech), and 15 clones were sequenced for each sample. Primers are listed in Table S5 .
6
Cloning and sequencing of METTL3 and METTL14
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SBPH genome in NCBI and an unpublished transcriptomic database (from sequences previously generated in our laboratory) were searched for orthologues of N. lugens genes of RNA methyltransferases METTL3 and METTL14 (Zhu et al., 2017 ) to design primers to amplify the CDS of LsMETTL3 and LsMETTL14 (Table S3 ). Total RNA was isolated from 15 to 20 SBPHs using the method described above, and the quality and concentration of total RNA were determined with the NanoDrop 2000 (Thermo Fisher Scientific). The extracted RNA (500 ng) was subsequently used for reverse transcription in a 20‐µl mixture as described in section 4.3 . The CDS of LsMETTL3 and LsMETTL14 was amplified by PCR using the CDS primers (Table S3 ), and all PCR products were gel purified and ligated into separate pEASY‐T5 Zero cloning vector according to the manufacturer's instructions (TransGen Biotech). These plasmids were then used to transform Escherichia coli DH5α cells. Cells containing the recombinant plasmid were selected using ampicillin (50 mg/ml), and the presence of the plasmid insert was verified by PCR. Subsequently, the CDS of LsMETTL3 and LsMETTL14 in the plasmids were sequenced by Sanger sequencing (Sangon). Finally, the recombinant plasmid DNA with the confirmed sequence was extracted using an Axyprep Plasmid Miniprep Kit (Axygen Biosciences).
7
CRISPR-Cas9 Editing of Human ERO1A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 20-nt guide sequences targeting human ERO1A were designed and cloned into the expression vector pSpCas9(BB)-2A-GFP (PX458) containing human codon optimized Cas9, the RNA components and GFP (a gift from Dr. Feng Zhang, Addgene plasmid # 48138). The guide sequence targeting exon 1 of human ERO1A is:
5′-CGGCTGGGGATTCTTGTTTG- 3′ (clone 10 and 13).
HeLa cells were transfected with pSpCas9(BB)-2A-GFP vector containing the single-guide RNAs (sgRNAs) by Viafect (Promega) for 48 h, and GFP-positive cells were single-cell-sorted and screened. Genomic DNA (gDNA) was purified from clones using the TransDirect Animal Tissue PCR Kit (TransGen), and the region surrounding the protospacer adjacent motif (PAM) was amplified using the following primers:
Forward: 5′- GATCGCTGAGAGGCAGGA- 3′.
Reverse: 5′- AGAAGCACCTCTGTGCCG- 3′.
PCR products were cloned into pEASY-T5 Zero Cloning Vector (TransGen). The InDels of individual alleles were determined by plasmid DNA purification and sequencing (Invitrogen).
5′-CGGCTGGGGATTCTTGTTTG- 3′ (clone 10 and 13).
HeLa cells were transfected with pSpCas9(BB)-2A-GFP vector containing the single-guide RNAs (sgRNAs) by Viafect (Promega) for 48 h, and GFP-positive cells were single-cell-sorted and screened. Genomic DNA (gDNA) was purified from clones using the TransDirect Animal Tissue PCR Kit (TransGen), and the region surrounding the protospacer adjacent motif (PAM) was amplified using the following primers:
Forward: 5′- GATCGCTGAGAGGCAGGA- 3′.
Reverse: 5′- AGAAGCACCTCTGTGCCG- 3′.
PCR products were cloned into pEASY-T5 Zero Cloning Vector (TransGen). The InDels of individual alleles were determined by plasmid DNA purification and sequencing (Invitrogen).
8
5'RACE of HMOX1 transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5′RACE assays were performed to amplify the 5′-end of HMOX1 transcripts from 4A+, hmox1, and ho1su1 as recommended by a previously published method (Scotto-Lavino et al., 2006 (link)). In brief, DNaseI-treated total RNA, primer G1 targeting exon2 of HMOX1, and M-MLV reverse transcriptase were used for the reverse transcription of the 5′-end of the HMOX1 mRNA. The product was ligated to poly(G) at the 5′-end and then used as a template for the 1st round of amplification with primers Qo, Qc, and gene-specific primer G2 targeting exon2 of HMOX1. The PCR product was diluted 10-fold and used as a template for further PCR amplification (2nd round) with primer Qi and gene-specific primer G3 spanning exon1 and exon2 of HMOX1. The final PCR product was analyzed using agarose gel electrophoresis and sequenced. Alternatively, the final PCR product was ligated into the pEASY-T5 Zero cloning vector (Transgen, Beijing, China) and introduced into competent E. coli Tans1-T1. Colonies that yielded PCR products larger than 250 bp when they were analyzed with M13F and M13R primers were used for plasmid extraction. Plasmids were sequenced to confirm they contained cDNA sequences derived from various types of HMOX1 transcripts from 4A+, hmox1, and ho1su1.
9
BmNPV Genome DNA Cloning and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified BmNPV genome DNA products were ligated into a pEASY-T5 Zero cloning vector (TransGen Biotech, Beijing, China) and sequenced using M13 primers. All primers used to detect the target gene mutation are presented in Supplementary Table S1 .
10
Genomic Sequence Amplification and KO Efficiency Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total 500 bp of target genomic sequence that was obtained from trachea and other tissues (tissues without trachea) of the BmMMP1-KO mutants were amplified by PCR. The purified DNA was ligated into a pEASY-T5 Zero cloning vector (TransGen, Beijing, China). The positive bacteria colonies were sent for sequencing using M13 primers and counted the knockout efficiencies. All the primers were listed in Supplementary Table S1 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!