The full-length mouse Pc4 cDNA (MC203765, Origene Technologies, Rockville, MD) and reverse tetracycline-controlled transactivator protein (rtTA; Clontech, Shiga, Japan) were PCR-amplified using the Pc4 open reading frame (ORF) and a plasmid containing rtTA, and subcloned into the pCR®8/GW/TOPO® (Invitrogen, Carlsbad, CA) Gateway recombinational cloning entry vector. The Pc4 ORF and rtTA sequence in pCR®8/GW/TOPO® (Invitrogen) was transferred to the CSII-EF-RfA-IRES2-Venus lentiviral vector (RIKEN, Ibaraki, Japan) by the Gateway® LR clonaseTM II (Invitrogen) reaction. A tetracycline (tet)-inducible lentivirus designated LV-tetO containing mouse Oct4, Sox2, Klf4 and c-Myc was obtained from Addgene (Cambridge, MA). Lentiviral vectors were produced by transient triple-plasmid transfection into 293FT cells (Invitrogen). For more details, see Supplementary Methods.
Pcr8 gw topo
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCR8/GW/TOPO is a cloning vector system designed for efficient and versatile cloning of PCR products. It provides a rapid and convenient way to clone PCR amplified DNA fragments for further analysis and manipulation.
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Lab products found in correlation
Lr clonase 2, by Thermo Fisher Scientific (10 mentions)
Lr clonase, by Thermo Fisher Scientific (9 mentions)
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (6 mentions)
Pentr d topo, by Thermo Fisher Scientific (5 mentions)
Gateway technology, by Thermo Fisher Scientific (4 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (4 mentions)
Lr clonase enzyme mix, by Thermo Fisher Scientific (3 mentions)
Gateway lr clonase 2, by Thermo Fisher Scientific (3 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (3 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Qiaquick spin column, by Qiagen (2 mentions)
Pyes dest52, by Thermo Fisher Scientific (2 mentions)
Pdest22, by Thermo Fisher Scientific (2 mentions)
Pdest17, by Thermo Fisher Scientific (2 mentions)
Plasmid midi kit, by Qiagen (2 mentions)
Lr clonase enzyme mix 2 kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Custom vector conversion kit, by Thermo Fisher Scientific (1 mentions)
104 protocols using pcr8 gw topo
1
Generation of Tetracycline-Inducible Lentiviral Vectors
2
Yeast Two-Hybrid Assay for Protein-Protein Interactions
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Mature OZ1 coding sequence (without the N-terminal predicted 33 aa transit peptide) was amplified using primer pair OZ1_100F and OZ1_R from cDNA and cloned into PCR8/GW/TOPO (Life Technologies, Carlsbad, CA). Mature RIP2 and RIP9 coding sequences were amplified using primer pairs RIP2_133F and RIP2_R, RIP9_175F and RIP9_R from A. thaliana cDNA, respectively. PCR products were first cloned into PCR8/GW/TOPO and then pGADT7GW and pGBKT7GW destination vectors through homologous recombination by LR clonaseII (Life Technologies, Carlsbad, CA). RIP1, RARE1, CRR28, OTP82, ORRM1 constructs produced for Y2H assays were previously described [7 (link)]. Empty vectors were used as negative controls. Two mating types of the PJ69–4 yeast strain, a and α, were used. Single transformants were obtained by transformation while double transformants were produced through mating. Yeast harboring testing pairs were grown in leucine and tryptophan deficient media overnight before they were diluted with water to OD600 0.5, 0.05, or 0.005. 10 μl of each dilution was spotted onto leucine-, tryptophan-, histidine-, adenine-deficient media plates. Growth results were collected after 3 days incubation at 30°C.
3
Cloning and Characterizing Wnt11b Variants
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Full-length cDNAs for wnt11b.L were amplified from wild-type or wnt11b−/− mutant oocyte total RNA using a high-fidelity polymerase (Q5, New England Biolabs). PCR products were cloned into pCR8/GW/TOPO (Invitrogen) and individual clones were verified by sequencing. Primer sequences are presented in Table S1 . Desired clones in the (correct) 5′L1-3′L2 orientation were inserted via recombination into a pCS2+ Gateway-converted vector (Custom Vector Conversion Kit; Invitrogen). Template DNAs for sense transcripts were prepared from wnt11b/pcs2+ plasmids by NotI digestion. Capped messenger RNA was synthesised using SP6 mMessage mMachine kits (Ambion). Eb3-gfp/pcs2+ RNA was similarly prepared as previously described (Olson et al., 2015 (link)). Mouse Lrp6 in pβ/RN3P was used as previously described (Kofron et al., 2007 (link)), prepared by SfiI/T3 digestion and transcription. RNAs for zebrafish dvl2-mcherry, rab5-gfp and rab11-gfp were gifts from D. Slusarski (The University of Iowa, IA, USA).
4
Cloning and Expression of Dclcyb1 Gene
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For Dclcyb1 expression, the complete Dclcyb1 coding sequence (DQ192190) was cloned into pCR®8/GW/TOPO (Invitrogen) using DcLcybF and DcLcybR (Supplementary Table S1 available at JXB online) and following the manufacturer’s instructions. Positive clones obtained by enzymatic digestion were sequenced by Macrogen Corp. (USA). Subsequently, pCR8/lcyb1 was recombined into pGWB2® (Curtis and Grossniklaus, 2003 (link)) to produce the pGWB2-Dclcyb1 expression vector (Moreno et al., 2013 (link)). Positive clones were analyzed through PCR and enzymatic digestion, and transformed into Agrobacterium tumefaciens (GV3101 strain).
5
Comprehensive RAP2.4 Sequence Analysis
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All sequences were extracted from the TAIR database [69 (link)]. The RAP2.4 cDNAs were re-sequenced after cloning them into pCR8/GW/TOPO (Invitrogen, Carlsbad, U.S.A.). Sequence alignments were performed online with CLUSTAL Ω [70 (link)] and MUSCLE [71 (link)]. Protein modelling was performed with SWISS-MODEL [33 (link)] and RasMol [35 (link)]. For comparison the models were overlaied with DeepView [33 (link)].
Gene expression intensity and transcript abundance co-regulation were analyzed on the Genevestigator platform [26 (link)], using all available data sets.
Gene expression intensity and transcript abundance co-regulation were analyzed on the Genevestigator platform [26 (link)], using all available data sets.
6
Gateway-Adapted Cloning for DNA Construction
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We followed the Gateway®-adapted cloning scheme developed by Margaret Phillips and colleagues
[40 (link)]. Briefly, a 300 to 400 base pair region was PCR amplified and TA-cloned (deoxythymidine, T, deoxyadenosine, A) into plasmid pCR/8GW/TOPO (Invitrogen, Grand Island, NY, USA) to generate an entry clone. The entry clone was then recombined with the destination vector pTrypRNAiGate. Final constructs were verified by restriction enzyme digestions and DNA sequencing.
[40 (link)]. Briefly, a 300 to 400 base pair region was PCR amplified and TA-cloned (deoxythymidine, T, deoxyadenosine, A) into plasmid pCR/8GW/TOPO (Invitrogen, Grand Island, NY, USA) to generate an entry clone. The entry clone was then recombined with the destination vector pTrypRNAiGate. Final constructs were verified by restriction enzyme digestions and DNA sequencing.
7
Cloning and Sequencing of Feverfew LTPs
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After getting the full length sequences of all LTPs Q5 High-Fidelity polymerase PCR was used for full length genes amplification. Feverfew RNA from developmental stage 4 was used to synthesize the cDNA according to manufacturer’s protocol (iScript™ cDNA Synthesis Kit, Bio-Rad). Full length sequences were amplified from feverfew cDNA. Primers are listed in Supplementary Table 1. All TpLTPs, except TpLTP3 were cloned into pCR™8/GW/TOPO® (Invitrogen™) entry vector by the addition of an adenosine overhang into their 3′ end post amplification. An overhang of CACC was added to the forward primer of TpLTP3 and was cloned into entry vector pENTR™/D-TOPO® (Invitrogen™). TpLTP 1–8 sequences have been deposited in the GeneBank under the accession numbers of MG550248 to MG550255.
8
Subcellular Localization of OsMYB7
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The full-length coding sequence of OsMYB7 subcloned into the entry vector pCR™8/GW/TOPO® (Invitrogen) was transferred into the Gateway-compatible plant destination vector pEarleyGate 104 (Earley et al., 2006 (link)) by the LR reaction. The resulting vector was introduced into onion (Allium cepa) epidermal cells by particle bombardment with a Biolistic PDS-1000/He instrument (Bio-Rad, Hercules, CA, USA). After bombardment, the onion epidermal layers were incubated on Murashige and Skoog (MS) medium (pH 5.7) for 16 h at 22°C in the dark. The nuclei were then stained with 300 nM of 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) in phosphate-buffered saline (PBS) for 3 min in darkness before observation. Fluorescence emission was analyzed using a Leica TCS SP8 X confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) with the following parameters: excitation at 458 nm and emission at 514 nm for YFP, and excitation at 405 nm and emission at 488 nm for DAPI.
9
High-Fidelity DNA Amplification and Cloning
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PfuUltra High-Fidelity DNA Polymerase (Stratagene) or EASYA DNA polymerase (Agilent) was used to amplify selected fragments (see above) by using isogenic genomic DNA from y; cn bw sp (73 (link)) as a template. The PCR products were confirmed by agarose gel analysis, purified by using the QIA-quick PCR Purification Kit (Qiagen). PCR fragment cloning was performed by adding three A-overhangs to the PCR products produced using the PfuUltra High-Fidelity DNA Polymerase (A-overhangs were not added to the products produced using the EasyA DNA Polymerase) with the addition of dATP and Taq polymerase in a 10-min incubation at 72 °C before Qiagen purification. The products (9.5 μL of each) were used in a TA TOPO cloning reaction with pCR8/GW/TOPO, as described by the manufacturer (Invitrogen). Cloning reactions were allowed to proceed for 30 min at room temperature, and then 2 μL of each reaction was used to transform Mach1 cells (Invitrogen). For each cloning reaction, two isolates were picked, purified, and confirmed by sequence verification.
10
Cloning and Expressing PROAtCAPE1 in Arabidopsis
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Total RNA extracted from Ler was utilized as the template for reverse transcription to generate cDNA. The cDNA was used as template for PCR using primers (Supplementary Table S2 ) corresponding to the 5′ and 3′ ends of the PROAtCAPE1 coding sequence. The amplified DNA fragment was cloned into pCR8/GW/TOPO (Invitrogen) according to the manufacturer’s protocol. The validated PROAtCAPE1 sequence was then cloned into pMDC32 (Curtis and Grossniklaus, 2003 (link)), a constitutive expression vector harbouring a dual 35S promoter, by site-specific recombination (LR clonase; Invitrogen). Similarly, for the generation of CAPE1oxCNYD transgenic plants, the PROAtCAPE1 sequence was recombined into pK7YWG2 (Karimi et al., 2002 (link)). For the generation of pPROAtCAPE1:GUS lines, the sequence upstream of the transcription start site of PROAtCAPE1 (2kb) was cloned into pCR8/GW/TOPO and then recombined into pMDC164 (Curtis and Grossniklaus, 2003 (link)). Transgenic Arabidopsis was generated via an Agrobacterium tumefaciens-mediated transformation system (Zhang et al., 2006 ).
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