Fragments of the human CYP 2C8 and 2J2 gene were cloned by using TOPO TA Cloning kit (Thermo Fisher Scientific, Waltham, MA). The forward primer (bases 869-888; 5’-CAA TCC TCG GGA CTT TAT CG-3’) and reverse primer (bases 1182-1163; 5’-GGA CAA GGT CAC TGT ATC TC-3’) for 2C8 were designed from the human 2C8 sequence (accession number NM_000770). The forward primer (bases 942-961; 5’ ACC GAG ACA ACT TGG ACA AC-3’) and reverse primer (bases 1347-1328; 5’-ATG CCC GCT TTC CTA TTG AG-3’) for 2J2 were designed from the human 2J2 sequence (accession number NM_000775). The CYP 2C8 and 2J2 fragments were amplified from aMHC-2C8 and aMHC-2J2 constructs. PCR was conducted for 40 cycles of denaturation (95°C, 45 seconds), annealing (61.6°C, 45 seconds), and extension (72°C, 1 minute). The resulting 314bp (2C8) and 406bp (2J2) products were subcloned into the pCR2.1-TOPO vector (Invitrogen) using the TOPO TA Cloning kit according to the manufacturer's instruction. The pCR2.1-TOPO vector contains only one promoter (T7), therefore, sense and antisense probes required two separate vectors with the respective inserts in the correct orientation for sense and antisense synthesis. The vectors containing the inserts were then sequenced to confirm the direction for sense and antisense probe synthesis.
Topo ta cloning kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Japan, France, Italy, Switzerland, China, Singapore, Canada
The TOPO TA Cloning Kit is a fast and efficient system for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector. It provides a simple, one-step cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector.
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Lab products found in correlation
Qiaprep spin miniprep kit, by Qiagen (57 mentions)
Qiaquick gel extraction kit, by Qiagen (46 mentions)
Qiaquick pcr purification kit, by Qiagen (30 mentions)
Epitect bisulfite kit, by Qiagen (28 mentions)
Rneasy mini kit, by Qiagen (24 mentions)
Ez dna methylation gold kit, by Zymo Research (20 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (18 mentions)
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (15 mentions)
Qiaamp dna mini kit, by Qiagen (15 mentions)
Dneasy blood and tissue kit, by Qiagen (13 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (12 mentions)
Firstchoice rlm race kit, by Thermo Fisher Scientific (12 mentions)
Dneasy blood tissue kit, by Qiagen (11 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (11 mentions)
Ez dna methylation direct kit, by Zymo Research (11 mentions)
Purelink quick plasmid miniprep kit, by Thermo Fisher Scientific (9 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (9 mentions)
Ez dna methylation kit, by Zymo Research (9 mentions)
Wizard sv gel and pcr clean up system, by Promega (8 mentions)
986 protocols using topo ta cloning kit
1
Cloning and Sequencing of CYP 2C8 and 2J2 Genes
2
Cloning and Sequencing of nifH Gene
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Depending on the band intensity, 3–4 µL of the gel-purified fragment of the PCR-amplified nifH gene (approximately 360 bp) was ligated into the plasmid vector (pCR® 2.1-TOPO® vector, 3.9 kb) using a TOPO® TA Cloning Kit (Invitrogen, Waltham, MA, USA, 2006) according to the manufacturer’s instruction. The heat shock method of transformation on One Shot® Chemically Competent E. coli cells (TOPO10) and the screening of transformed cells were carried out as described in TOPO TA Cloning Kit (Invitrogen, 2006).
Cloned PCR products with the right insert were screened by PCR amplification using M13 primers directly from overnight cultured transformed E. coli cells, and by running the amplified PCR products on agarose gel (3% w/v) electrophoresis. Clones with the right insert were sequenced using primer sets, nifH1 and nifH2, from both directions.
Cloned PCR products with the right insert were screened by PCR amplification using M13 primers directly from overnight cultured transformed E. coli cells, and by running the amplified PCR products on agarose gel (3% w/v) electrophoresis. Clones with the right insert were sequenced using primer sets, nifH1 and nifH2, from both directions.
3
Whole Mount RNA In Situ Hybridization in Zebrafish
4
TOPO TA Cloning and Colony PCR
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PCR products were cloned into pCR® 2.1-TOPO® vector, TOPO® TA Cloning® Kit, (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and using Mach1-T1 competent bacteria. The colony PCR with Universal M13 primers was performed in 80 colonies with Qiagen® Multiplex PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. M13 forward and reverse primers were provided by the TOPO® TA Cloning® Kit, (Invitrogene, Carlsbad, CA, USA) and used in the reaction at 0.4 μm (primers and PCR conditions are described in Supplemental Table S2 ). Amplification was confirmed by electrophoresis of the products in a 2% agarose gel, and the selected amplified products were sequenced using the primers and conditions described in Supplemental Table S2 .
5
Cloning and Expression of HvYS1 Gene
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A full-length cDNA of HvYS1 (DNA Data Bank of Japan (DDBJ) under the accession number AB214183) was amplified with the forward 5′-GCTCTAGAATGGACATCGTCGCC-3′ and the reverse 5′-CCCAAGCTTTTAGGCAGCAGGTAG-3′ primers, which were subcloned to the PERII-TOPO vector with a TOPO-TA cloning kit (Life Technologies Corporation, Grand Island, NY, USA). The HvYS1 cDNA was inserted into the Mac-1 promoter [33 (link)] and the mannopine synthase (mas) terminator [34 (link)] in the sense orientation. The constructed expression cassette was inserted into a binary vector, pBinPLUS [35 (link)], to produce the plasmid Mac-HvYS1-mas-pBinPlus shown in supporting information (S1 Fig. ).
6
Arabidopsis HSP70 ORF Cloning
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The cDNA obtained from Arabidopsis leaves treated at 40°C for 30 min (treatment B) was used as template for the amplification of the full length inducible HSP70 ORF using the proofreading Expand PCR System (Roche). Primers were as follows: AtHSP70F: 5'TAATGGCGGGTAAGGTGAA; AtHSP70R: 5'GCCAAAAGGCTTAATCAACTTC. The amplified product was cloned using the TOPO TA Cloning Kit (Life-Technology) and sequenced. The cloned fragment was then amplified by PCR with primers containing the XbaI and BamHI recognition sequences to the 5’ and 3’ end respectively (forward primer: 5’tctagaATGGCGGGTAAAGG; reverse primer: 5’ggatccGCCAAAAGGCTTA, restriction sites in small case) and the gene was cloned under the transcriptional control of the CaMV35S promoter and the Nos terminator in a Bin19-derived [23 (link)] plant transformation vector, thus obtaining the pJAZZ-HSP70 vector (Fig 1 ). The correctness of the HSP70 ORF was checked by sequencing. The plasmid was introduced into A. tumefaciens strains LBA4404 and AGL1 by electroporation
7
TA Cloning and Sanger Sequencing of PCR Products
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Positive PCR products were cloned by TA cloning (TOPO TA cloning kit Life Technologies, Carlsbad, CA, USA; cat. no. 45-0641) and the ligation mix was transformed in competent E. coli DH5 cells. Plasmid DNA was purified using the NucleoSpin Plasmid prep kit according to the manufacturer’s instructions (Machery-Nagel, Düren, Germany; cat. no. 740588.250) and sequenced with the M13 reverse primer (5′-CAGGAAACAGCTATGAC-3′) using the BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFisher Scientific, Waltham, MA, USA; cat. no. 4337455) and the ABI 35000xL (Applied Biosystems, Foster City, CA, USA). The obtained sequences were compared to reference sequences deposited in GenBank (GenBank accession numbers (MZ081042-MZ081051). Sequence alignments were performed with the Clustal Omega Multiple Sequence Alignment algorithm [43 (link)].
8
Cloning and Sequencing of p13 Region
9
T Helper Cell Gene Expression Analysis
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T helper cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS), lysed with RLT buffer and RNA was extracted (RNAeasy mini kit Qiagen, Gaithersburg, MD) per manufacturer’s instructions. cDNA was synthesized (First-Strand cDNA synthesis Origene, Rockville, MD) per manufacturer’s instructions. Quantitative PCR (qPCR) was performed on a 7300 Real Time PCR System (Applied BioSystems, Foster City, CA). Primers were designed using Integrated DNA technology website (http://www.idtdna.com/Primerquest/Home/Index ) with sequences from Genebank accession numbers (Table 1 ). Changes in gene expression were calculated by the ΔΔCT method[47 (link)] and depicted as fold change in gene expression compared to control.
To confirm the specificity of our primers, qPCR products were run on a 2% agarose gel and product size as well as the presence of non-specific products were determined. IL17A and RORc qPCR products were further cloned into 2.1 PCR product vector per manufacturer’s instructions (TOPO-TA Cloning Kit, Life technologies). Plasmids were then transfected into OneShot® cells and positive colonies selected and expanded overnight prior to plasmid isolation (Wizard® Plus Minipreps DNA Purification System, Promega, Madison, WI) and sequencing by an automated sequencer (Nucleics, Davis, CA).
To confirm the specificity of our primers, qPCR products were run on a 2% agarose gel and product size as well as the presence of non-specific products were determined. IL17A and RORc qPCR products were further cloned into 2.1 PCR product vector per manufacturer’s instructions (TOPO-TA Cloning Kit, Life technologies). Plasmids were then transfected into OneShot® cells and positive colonies selected and expanded overnight prior to plasmid isolation (Wizard® Plus Minipreps DNA Purification System, Promega, Madison, WI) and sequencing by an automated sequencer (Nucleics, Davis, CA).
10
Quasi-species Analysis of Viral Capsid Sequences
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To assess quasi-species at VP1-98 and VP-145, partial capsid cDNA (about 700 nt covering VP3-214 to VP1-210) was amplified by VP1-1 and VP1-3R primers directly from tissue or clinical samples and cloned into a pCR-TOPO vector using TOPO-TA cloning kit (Life technologies, Carlsbad, CA) according to the manufacturer’s instructions. Resultant colonies were randomly selected and plasmids were purified using QIAprep Miniprep kits (QIAGEN, Hilden, Germany). In total, forty-four independent plasmids carrying viral cDNA from 7 postmortem samples (3 CNS, 3 non-CNS, and 1 PBMC samples) from 02363-KE- or 02363-EG-inoculated monkeys were sequenced. Similarly, thirty independent plasmids derived from 4 clinical samples (2 rectal and 2 throat swab samples) from two 02363-KE-inoculated monkeys were analyzed. Viral quasi-species variants were identified by nucleotide sequence alignment of plasmid clones derived from each clinical or tissue sample.
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