The gene-specific probes were cloned by PCR into a pGEM-T Easy TA cloning vector (Promega) using the primers listed in Table S2 . Antisense probes were synthesized by in vitro transcription using DIG RNA Labeling Kit (SP6/T7) (Roche Applied Science). ISH was performed as previously described [29 (link)].
Pgem t easy ta cloning vector
Manufactured by Promega
Sourced in United States
The pGEM-T Easy TA cloning vector is a linear plasmid designed for the direct cloning of PCR products. It contains a T7 RNA polymerase promoter, a multiple cloning site, and a lacZ gene for blue-white color screening of recombinant clones.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling kit sp6 t7, by Roche (2 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
In fusion, by Takara Bio (1 mentions)
Kod fx neo polymerase, by Toyobo (1 mentions)
Pri 201 an vector, by Takara Bio (1 mentions)
Plvx puro vector, by Takara Bio (1 mentions)
Takara ex taq dna polymerase, by Takara Bio (1 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Purelink plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Seqman, by DNASTAR (1 mentions)
Megalign, by DNASTAR (1 mentions)
Editseq, by DNASTAR (1 mentions)
3730 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
I taq dna polymerase, by iNtRON Biotechnology (1 mentions)
Dig rna labelling kit sp6 t7, by Roche (1 mentions)
Random primer dna labeling kit, by Takara Bio (1 mentions)
10 protocols using pgem t easy ta cloning vector
1
Gene-Specific Probe Synthesis for ISH
2
Heterologous Expression of Terpene Synthases
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RiRZS1, NCBI Accession no. JN166691, was obtained as previously described by Koeduka et al. (2011) (link). RpBAS, NCBI Accession no. AF326911, was synthesized from GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). For heterologous expression of RpBAS and RiRZS1 in tobacco plants, the full-length cDNAs were amplified with KOD FX Neo polymerase (Toyobo Co. Ltd., Osaka, Japan) using gene-specific primers (Supplementary Table 1 ). The resulting PCR products of RpBAS and RiRZS1 containing flanking NdeI and XhoI or SalI restriction sites were cloned into the NdeI and SalI-digested pRI201-AN vector (Takara Bio USA, Mountain View, CA, USA) independently via the intermediate pGEM-T easy TA-cloning vector (Promega, Tokyo, Japan). The RpBAS expression cassette containing the CaMV35S promoter with Arabidopsis alcohol dehydrogenase 5′-untranslated region and heat shock protein terminator was amplified using PCR and cloned into the pro35S:RiRZS1 plasmid behind the RiRZS1 expression cassette by InFusion (Takara Bio USA, Mountain View, CA, USA), according to manufacturer's instructions. The resulting vector carrying pro35S:RiRZS1-pro35S:RpBAS was transformed into Agrobacterium tumefaciens LBA4404 using electroporation.
3
Cloning and Expression of Heat-Stable Proteins
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The genes for screened heat-stable proteins including Snz1, Wos2, NTF2, and cofilin were cloned by PCR using S. pombe cDNA as a template. Primers for the cloning of heat-stable protein genes contained an NdeI site (underlined) in the forward primer and BamHI or EcoRI site (underlined) in the reverse primer: Snz-forward, 5'-AAA CAT ATG TCT GCC GAA ATT AAG-3' and Snz-reverse, 5'-AAA GGA TCC TTA CCA CCC GCG AGT-3'; Wos2-forward, 5'-AAA CAT ATG AGT TTA AAT ACA CAA-3' and Wos2-reverse, 5'-AAA GGA TCC TTA CTC TTT CTT TTC GTT-3'; NTF2-forward, 5'-AAA CAT ATG GCT GAC TAT AAT-3' and NTF2-reverse, 5'-AAA GAA TTC TCA ACC ATA GTT-3'; Cofilin-forward, 5'-AAA CAT ATG TCT TTT TCA GGT GTC-3' and Cofilin-reverse, 5'-AAA GGA TCC TTA CTT ACG AGT AAC-3'. The PCR reaction mixture contained 100 ng of S. pombe cDNA, 20 pmole of both forward and reverse primers, and 5× Taq-PCR Mix (Genotech Ltd., Daejeon, Korea). The purified PCR product was cloned into pGEM-T Easy TA cloning vector (Promega, Madison, WI, USA). The sequence of genes was confirmed by Solgent (Daejeon, Korea).
The PCR products containing genes of heat-stable proteins were expressed using pET17b system in E. coli BL21(DE3) with induction by 0.4 mM isopropyl β-D-1-thiogalactopyranoside. The overexpressed E. coli cells were harvested by centrifugation and stored at -20℃ until used.
The PCR products containing genes of heat-stable proteins were expressed using pET17b system in E. coli BL21(DE3) with induction by 0.4 mM isopropyl β-D-1-thiogalactopyranoside. The overexpressed E. coli cells were harvested by centrifugation and stored at -20℃ until used.
4
Cloning and Tagging of Androgen Receptor Variants
5
Bacterial Identification by Colony PCR
6
Cloning and Sequencing DENV2 E-gene
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E-gene 346-bp fragments from DENV2-positive samples were cloned into a pGEM-T Easy TA cloning vector (Promega, Madison, WI, USA) and chemically transformed into DH5α Escherichia coli. White transformed colonies were randomly selected from LB/Amp/IPTG/X-gal agar plates and subjected to colony PCR to check for the presence of an insert. Using a Purelink Plasmid miniprep kit (Invitrogen), plasmid DNA was then isolated from overnight cultures of single positive clones grown in Luria–Bertani broth containing 100 µg ml−1 ampicillin. Six to 10 clones from each human sample and 14 clones from each mosquito sample were sequenced using M13 universal primers. The DENV2 E gene sequences generated from both humans and mosquitoes were submitted to the DNA Data Bank of Japan (DDBJ) under accession numbers LC030023–LC030105.
7
Genomic DNA Extraction and PCR Amplification
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Genomic DNA was isolated from well-expanded leaves from the field grown plants following the protocol of Rogers and Bendich [22 ]. All PCR amplifications were carried out in a 20 μl reaction volume containing 50 ng of template DNA, 800 μM of dNTPs, 2 mM of MgCl2, 10 pmol each of forward and reverse primers and enzyme Taq polymerase (1 Unit, i-Taq DNA polymerase, Intron Biotechnology, Inc.). The PCR profile included initial denaturation at 94°C for 5 min followed by 36 cycles of 94°C for 30 sec, annealing at 62°C for 30 sec and 72°C for 1 min 30 sec and a final extension at 72°C for 12 min. For the purpose of sequencing, amplified fragments were cloned in the pGEM-T Easy TA cloning vector (Promega, Madison, USA). Sequencing was done by capillary electrophoresis on Applied Biosystems 3730 Genetic Analyzer. Sequencing of each sample was done in a minimum of 3 independent replicates to remove chances of any PCR related error. Sequence files were assembled and analysed using EditSeq, SeqMan and MegAlign software packages of DNAStar (DNAStar Inc.). For mapping, marker genotyping data of the mapping population were added to the existing map of B. juncea [21 (link)] using the program JoinMap version 4.0 [23 ]. Genome walk was performed using PCR genomic libraries based on the method described by GenomeWalker Universal Kit (Clontech Ltd.).
8
In Situ Hybridization Protocol
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In situ hybridization was performed as previously described [69 (link)]. Using the primers listed in Table 1 , the gene-specific probes were cloned by PCR into a pGEM-T Easy TA cloning vector (Promega). In vitro transcription was used to create antisense probes using the DIG RNA Labeling Kit (SP6/T7) (Roche Applied Science, Penzberg, Germany).
9
Gene-specific probe cloning and in situ hybridization
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The gene-specific probes were cloned by PCR into a pGEM®-T Easy TA cloning vector (Promega, Madison, WI USA) using the primers listed in Supplementary Table S2 . Antisense probes were synthesized by in vitro transcription using the DIG RNA Labelling Kit (SP6/T7) (Roche Applied Science, Mannheim, Germany). ISH was performed as described previously [34 (link)].
10
Cloning and Characterization of Glucose Transporter CL353
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The glucose transporter candidate CL353 was found in a C. sinensis expressed sequence tag (EST) raw database (http://grc.kribb.re.kr/pipeline2). This gene matched a protein sequence (protein id GAA56139.1) in the National Center for Biotechnology Information (NCBI) nucleotide database. The nucleotide sequence of CL353 was prepared using chromosomal nucleotide sequence DF144168.1. The PCR primers for probe DNA fragment preparation were forward, 5′-TTA ATA CGG AAA CTC TAT GGC-3′ (corresponding to nucleotide 602-622 of the coding region), and reverse, 5′-TAT TCA ATA TAT TGG CTC GGG-3′ (corresponding to nucleotide 972-992 of the coding region). Five micrograms of total RNA prepared from adult C. sinensis was transcribed and used as a template for subsequent PCR. PCR was performed according to the following protocol: predenaturation at 94 °C for 10 min followed by 35 cycles (94 °C for 60 s, 55 °C for 60 s, and 72 °C for 90 s) and final extension at 72 °C for 10 min. The PCR product obtained was subcloned into pGEM-T Easy TA cloning vector (Promega, USA), and its nucleotide sequence was confirmed. The [ 32 P]dCTP-labeled probe was synthesized using a random primer DNA labeling kit (TaKaRa, Japan) from the PCR clone and used as probe for screening of the C. sinensis cDNA library and for Northern blot analysis.
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