Total DNA of R. sativus was isolated from radish seedlings using the cetyltrimethylammonium bromide (CTAB) method [42 (link)]. To amplify the fragments of radish CLE genes, PCR was performed with primers designed for the conserve regions of corresponding genes of Arabidopsis thaliana and Brassica rapa using a VectorNTI Advance_10 (Invitrogen, United States) program (Additional file 4 : Table S2). PCR-products were separated by electrophoresis in 1 % agarose gel containing ethidium bromide (0.1 %). Target fragments were isolated from the gel using a Cleanup Mini Kit (Evrogen, Russia) according to the manufacturer’s instructions and cloned into the pAL-TA vector (Evrogen, Russia). Transformation of chemically competent cells of Escherichia coli strain DH5α was carried outaccording to the protocol described in [43 (link)]. Transformants were selected on solidified LB medium containing 100 mg/L ampicillin and X-Gal. Plasmid DNA of selected transformant clones was isolated by Plasmid Miniprep Kit (Evrogen, Russia) and sequenced in SPbU Research Park, Center of Molecular and Cell Technologies. Sequence aligning of Arabidopsis and radish genes was performed using the AlignX program of VectorNTI Advance_10 (Invitrogen) software.
Vector nti advance 10
Manufactured by Thermo Fisher Scientific
Sourced in United States
Vector NTI Advance 10 is a software suite designed for molecular biology analysis and visualization. It provides tools for sequence assembly, alignment, primer design, and in-silico cloning.
Automatically generated - may contain errors
Lab products found in correlation
Hotstartaq dna polymerase, by Qiagen (13 mentions)
Wizard 96 magnetic dna plant system kit, by Promega (13 mentions)
Mm00450960 m1, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna 2, by Macherey-Nagel (2 mentions)
Plasmid miniprep kit, by Evrogen (1 mentions)
Cleanup mini kit, by Evrogen (1 mentions)
Pal ta vector, by Evrogen (1 mentions)
Chip it express, by Active Motif (1 mentions)
Gentra puregene kit, by Qiagen (1 mentions)
Amplitaq gold, by Thermo Fisher Scientific (1 mentions)
Methyl primer express v1, by Thermo Fisher Scientific (1 mentions)
Ez 96 dna methylation kit d5004, by Zymo Research (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Lightcycler lc480, by Roche (1 mentions)
Netprimer, by Premier Biosoft (1 mentions)
Sequence viewer 7, by Qiagen (1 mentions)
Transscript one step gdna removal and cdna synthesis supermix kit, by Transgene (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Evagreen, by Biotium (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
41 protocols using vector nti advance 10
1
Isolation and Characterization of Radish CLE Genes
2
Clonal Plant Leaf DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
Leaf tissue is collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA is extracted using a Wizard® 96 Magnetic DNA Plant System kit (Promega, U.S. Pat. Nos. 6,027,945 & 6,368,800) as directed by the manufacturer. Isolated DNA is PCR amplified using the appropriate forward and reverse primer.
PCR amplification is performed using Hotstar Taq DNA Polymerase (Qiagen) using touchdown thermocycling program as follows: 96° C. for 15 min, followed by 35 cycles (96° C., 30 sec; 58° C.-0.2° C. per cycle, 30 sec; 72° C., 3 min and 30 sec), 10 min at 72° C. PCR products are verified for concentration and fragment size via agarose gel electrophoresis. Dephosphorylated PCR products are analyzed by direct sequence using the PCR primers (DNA Landmarks, or Entelechon). Chromatogram trace files (.scf) are analyzed for mutation relative to the wild-type gene using Vector NTI Advance 10™ (Invitrogen). Based on sequence information, mutations are identified in several individuals. Sequence analysis is performed on the representative chromatograms and corresponding AlignX alignment with default settings and edited to call secondary peaks.
3
Clonal Plant Leaf Tissue DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 6
Leaf tissue was collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA was extracted using a Wizard® 96 Magnetic DNA Plant System kit (Promega, U.S. Pat. No. 6,027,945 & 6,368,800) as directed by the manufacturer. Isolated DNA was PCR amplified using the appropriate forward and reverse primer.
PCR amplification was performed using Hotstar Taq DNA Polymerase (Qiagen) using touchdown thermocycling program as follows: 96° C. for 15 min, followed by 35 cycles (96° C., 30 sec; 58° C.-0.2° C. per cycle, 30 sec; 72° C., 3 min and 30 sec), 10 min at 72° C.
PCR products were verified for concentration and fragment size via agarose gel electrophoresis. Dephosphorylated PCR products were analyzed by direct sequence using the PCR primers (DNA Landmarks, or Entelechon). Chromatogram trace files (.scf) were analyzed for mutation relative to the wild-type gene using Vector NTI Advance 10™ (Invitrogen). Based on sequence information, mutations were identified in several individuals. Sequence analysis was performed on the representative chromatograms and corresponding AlignX alignment with default settings and edited to call secondary peaks.
4
Leaf Genomic DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
Leaf tissue is collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA is extracted using a Chloropure Nucleic acid extraction kit (Agencourt, U.S. Pat. Nos. 5,898,071; 5,705,628; 6,534,262) as directed by the manufacturer. Isolated DNA is PCR amplified using the appropriate forward and reverse primer. PCR amplification is performed using LongAmp HotStart Taq DNA Polymerase Mix (New England Biolabs) using thermocycling program as follows: 94° C. for 30 sec, followed by 35 cycles (94° C., 30 sec; 54° C., 30 sec; 65° C., 300 sec), 10 min at 65° C.
PCR products are verified for concentration and fragment size via agarose gel electrophoresis. Dephosphorylated PCR products are analyzed by direct sequence using the PCR primers (Genewiz or GenScript). Chromatogram trace files (.scf) are analyzed for mutation relative to the wild-type gene using Sequencher (Gene Codes) or Vector NTI Advance 10™ (Invitrogen). Based on sequence information, mutations are identified in several individuals. Sequence analysis is performed on the representative chromatograms and corresponding alignment with default settings and edited to call secondary peaks.
5
Identifying p53 Repressive Response Elements in ENO3 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used UCSC Genome Browser (http://genome.ucsc.edu/ ) to obtain the human ENO3 genomic sequence. The potential p53 repressive response elements (p53RREs), which are consisting of four canonical p53-binding sites (5’-RRRCW-3′) arranged head to tail and matching >90%, were identified by Vector NTI Advance 10 software (Invitrogen).
ChIP assay was performed with ChIP-IT Express (Active Motif) following the manufacturer's protocol. A-204 cells were fixed with 1% formaldehyde on a shaking platform for 10 min at room temperature following by sonication to shear the chromatin. Samples were immunoprecipitated with mouse monoclonal anti-p53 antibody or control mouse IgG serum (10 μg/ml) for PCR amplification. CD44 served as the positive p53RRE control [21 (link)].
Sequences of RT-PCR primer pairs for the putative p53RREs:
ENO3-RRE1 (Foward:5′-CAGGCAATGTCTGGATCACCG-3′, Reverse:5′-CTGACTGCGAAGAAACCCAAAG-3′)
ENO3-RRE2 (Foward:5′-CCTGTCTAAATTCGTTTCCTGTCC-3′, Reverse:5′-CACCCCAGGATTACATTCCC-3′)
ENO3-RRE3 (Foward:5′-TTGACCTTTGGTAAGGGGGC-3′, Reverse:5′-AGCCAATACCATGCTCACCC-3′)
CD44-RRE (Foward:5′-TTTACGGTTCGGTCATCCTC-3′, Reverse:5′-TGCTCTGCTGAGGCTGTAAA-3′)
ChIP assay was performed with ChIP-IT Express (Active Motif) following the manufacturer's protocol. A-204 cells were fixed with 1% formaldehyde on a shaking platform for 10 min at room temperature following by sonication to shear the chromatin. Samples were immunoprecipitated with mouse monoclonal anti-p53 antibody or control mouse IgG serum (10 μg/ml) for PCR amplification. CD44 served as the positive p53RRE control [21 (link)].
Sequences of RT-PCR primer pairs for the putative p53RREs:
ENO3-RRE1 (Foward:5′-CAGGCAATGTCTGGATCACCG-3′, Reverse:5′-CTGACTGCGAAGAAACCCAAAG-3′)
ENO3-RRE2 (Foward:5′-CCTGTCTAAATTCGTTTCCTGTCC-3′, Reverse:5′-CACCCCAGGATTACATTCCC-3′)
ENO3-RRE3 (Foward:5′-TTGACCTTTGGTAAGGGGGC-3′, Reverse:5′-AGCCAATACCATGCTCACCC-3′)
CD44-RRE (Foward:5′-TTTACGGTTCGGTCATCCTC-3′, Reverse:5′-TGCTCTGCTGAGGCTGTAAA-3′)
6
Genetic Mapping of slc1 Mutant in Rice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The heterozygous slc1 plants were crossed with Oryza sativa subsp. indica ‘Jinhui 1’ for genetic analysis. The F1 seeds were harvested from each individual plant, and mutants segregating in the F2 population were selected for gene mapping. Sequence polymorphisms between Oryza sativa subsp. japonica ‘Nipponbare’ and subsp. indica ‘93–11’ were used to develop insertion/deletion and simple sequence repeat molecular markers (Shen et al. 2004 (link)). Gene annotation and primer design for DNA and cDNA sequencing were performed on the basis of information obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ ) database and Gramene (http://gramene.org/ ) databases. Multiple sequence alignment was performed with Vector NTI Advance 10 (Invitrogen, USA; http://www.invitrogen.com/ ). All primers used in this study are listed in Supplementary Table S2 .
7
Bioinformatic Characterization of Canine mda-7
8
Determination of p16 Promoter Methylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from BALB/c mouse thymus using the Gentra Puregene kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Determination of Percent Methylated Cytosine Genomic DNA (300 ng) was treated with sodium bisulfate using the EZ-96 DNA Methylation Kit D5004 (Zymo Research, Orange, CA) according to the manufacturer's protocol. The final bisulfite-treated DNA was eluted in 40 ml M-Elution Buffer. Primers for amplifying the upstream p16 CpG island using bisulfite-treated DNA were designed using Methyl Primer Express v1.0 (Applied Biosystems, Foster City, CA) and Vector NTI Advance 10 (Invitrogen, Carlsbad, CA). Amplification was performed with 1 µl bisulfite-treated DNA, 1 µM of each primer (primer : 5′-GTTAAAGGGTGATTAGGTATGG-3′ and :5′-ACCCCAACTTCCAACAATAC- 3′ , 250 µM each of dATP, dCTP, dGTP, and TTP, 50 µM KCl, 4 µM MgCl2, 0.625 U AmpliTaq Gold (Applied Biosystems), and 10 µM Tris-HCl (pH 8.3) in 50 µl. Amplification consisted of 5 min at 95°C, 40 cycles of 15 sat 95°C, 15 s at 52°C, and 30 s at 72°C, followed by a final elongation step at 72°C for 7 min.
9
Leaf DNA Extraction and Mutation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
Leaf tissue was collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA was extracted using a Wizard® 96 Magnetic DNA Plant System kit (Promega, U.S. Pat. Nos. 6,027,945 & 6,368,800) as directed by the manufacturer. Isolated DNA was PCR amplified using the appropriate forward and reverse primer.
PCR amplification was performed using Hotstar Taq DNA Polymerase (Qiagen) using touchdown thermocycling program as follows: 96° C. for 15 min, followed by 35 cycles (96° C., 30 sec; 58° C.-0.2° C. per cycle, 30 sec; 72° C., 3 min and 30 sec), 10 min at 72° C. PCR products were verified for concentration and fragment size via agarose gel electrophoresis. Dephosphorylated PCR products were analyzed by direct sequence using the PCR primers (DNA Landmarks, or Entelechon). Chromatogram trace files (.scf) were analyzed for mutation relative to the wild-type gene using Vector NTI Advance 10™ (Invitrogen). Based on sequence information, mutations were identified in several individuals. Sequence analysis was performed on the representative chromatograms and corresponding AlignX alignment with default settings and edited to call secondary peaks.
10
Sequencing and Expression Analysis of ZmVDE1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two primers that spanned most of the gene were used in conjunction with two additional sets of primers to sequence ZmVDE1 in 89 maize lines and 44 teosinte entries (Supplementary Table S2 ). A 30-μl aliquot of the resulting PCR product from each line was sequenced directly using an ABI3730 sequencer. The sequences were assembled using ContigExpress in Vector NTI Advance 10 (Invitrogen), aligned using MUSCLE (Edgar, 2004 (link)) and manually corrected with BioEdit (Hall, 1999 ). Additionally, to determine the expression level of ZmVDE1, 557,955 polymorphic sites with a minor allele frequency (MAF) ≥0.05 and missing rate <25% were generated by RNA-Sequencing maize kernels collected 15 days after pollination (DAP) from 368 maize inbred lines (Fu J.J. et al., 2013 (link)). Using the expression levels of ZmVDE1, we looked for expression QTL and analyzed their correlations with carotenoid content. Subsequently, >120,000 single nucleotide polymorphisms (SNPs) distributed across chromosome 2 in the 368 maize lines were used for testing whether there were selective sweeps around ZmVDE1.
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!