The tobacco BY-2 evacuolated protoplast lysate (BYL) was prepared as previously described (Komoda et al. 2004 (link); Ishibashi et al. 2006 ) and the membrane fraction was preserved. DRB7.1, DRB4, DRB2, RTL1, and RTL2 coding sequences were amplified by PCR and cloned into pSP64-poly(A) vector (Promega). HA/Flag epitopes were added during PCR using primers with overhangs encoding tag sequences. Primers are listed in Supplemental Table T1 . Plasmids were linearized and mRNAs were in vitro transcribed using AmpliCap SP6 High Yield Message Maker Kit (Cellscript). In vitro translation was carried out as previously described (Ishibashi et al. 2006 ). Briefly, mRNAs (0.05 µg/µL or 120 nM) were mixed in BYL with 10× translation substrate buffer (7.5 mM ATP, 1 mM GTP, 250 mM creatine phosphate, 0.8 mM spermine, Amino Acid Mixture [Promega]), RiboLock RNase inhibitor (Thermo Scientific), creatine kinase 10mg/mL (Roche) and Translation Reaction (TR) buffer (30 mM HEPES-KOH, 80 mM KOAc, 1.8 mM Mg(OAc)2, 2 mM DTT, pH 7.4). Mixtures were incubated 1–2 h at room temperature. One species of mRNA was translated per reaction.
Psp64 poly a vector
Manufactured by Promega
Sourced in United States
The PSP64-poly(A) vector is a plasmid for expressing recombinant proteins in mammalian cells. It contains a polylinker region for cloning and a poly(A) signal sequence for mRNA stability and processing.
Automatically generated - may contain errors
Lab products found in correlation
Amplicap sp6 high yield message maker kit, by CellScript (2 mentions)
Amino acid mixture, by Promega (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti myc antibody 9e10, by Santa Cruz Biotechnology (1 mentions)
Amplicap sp6 high yield message maker kit, by Illumina (1 mentions)
Supermix scintillation cocktail, by PerkinElmer (1 mentions)
Tnt sp6 coupled reticulocyte lysate system, by Promega (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
Scriptcap m7g capping system, by CellScript (1 mentions)
Sp6 scribe standard rna ivt kit, by CellScript (1 mentions)
Rnase inhibitor, by Transgene (1 mentions)
Geneart strings, by Thermo Fisher Scientific (1 mentions)
Tnt sp6 high yield wheat germ protein expression system, by Promega (1 mentions)
7 protocols using psp64 poly a vector
1
In Vitro Translation of Plant Proteins
2
Myc-nedd8 Expression in Zebrafish
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Myc-nedd8 and GFP were subcloned into Psp64 poly (A) vector (Promega). AmpliCap SP6 High Yield message maker kit (Epicenter) was used for capped mRNA synthesis. Myc-nedd8 and GFP mRNA were synthesized and injected into zebrafish embryos at one-cell stage (400 pg/per embryo). To confirm expression of injected mRNAs, the embryos injected with Myc-nedd8 mRNA for 3 days were harvested and the expression of Myc-nedd8 was confirmed by Western blot using anti-Myc antibody (9E10, Santa Cruz).
3
Quantitative Assay for Autoantibodies
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Recombinant GAD65 and IA-2 were labeled with 35S-methionine (PerkinElmer, Waltham, MA, USA) by in vitro coupled transcription and translation using the TNT SP6 coupled reticulocyte lysate system (Promega, Madison, WI, USA) as described [14 (link)]. Full length cDNA coding for human GAD65 in the pTNT vector (Promega) (pThGAD65) or the intracellular domain (amino acids 606-979) of IA-2 in the pSP64 Poly(A) vector (Promega) (IA-2ic) were used as templates [15 (link)]. GADA and IA-2A were analyzed in a radioligand binding assay (RBA) [14 (link)]. Duplicate samples were incubated with radio-labeled antigen. The samples were transferred to filtration plates (Millipore, Solna, Sweden) and IgG antibodies precipitated with Protein A Sepharose (Zymed Laboratories Inc, San Francisco, CA, USA). After washing to remove all unbound antigen supermix scintillation cocktail (Perkin Elmer) was added and the radioactivity counted in a Wallac Microbeta Trilux system (Perkin Elmer). GADA and IA2A levels were expressed as units per mL (U/mL) derived from the WHO standard 97/550. GADA levels >34 U/mL and IA-2A levels >5 U/ml were considered positive.
4
Radioactive GFP Fragment Synthesis
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A partial GFP fragment amplified by PCR using TI429 and TI431 (Supplemental Table S1 ) was cloned between SalI and BamHI sites in pSP64-poly(A) vector (Promega). Plasmid linearized by EcoRI was used as template for in vitro transcription with the SP6-scribe standard RNA IVT kit (Cellscript) in the presence of [α-32P]-CTP. The transcripts were capped with the ScriptCap m7G Capping System (Cellscript). The products were purified as described in “Preparation of double-stranded RNA.”
5
Synthesis of GFP RNA Transcripts
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The pSP64 polyA vector was purchased from Promega (USA). The GFP ORF was cloned from the vector pEGFP-N1 and inserted into the pSP64 polyA vector by HindIII to obtain the SP64-GFP plasmid. The primers used to clone GFP were as follows: antisense: 5′-GATCCACCGGTCGCCACCA-3′; sense: 5′-GTACAGCTCGTCCATGCCGAGAGT-3′. The plasmid SP64-GFP was separately digested with SacI or EcoRI to generate two different types of linear transcriptional templates. Two types of GFP RNA, without or with a 30-nt poly (A) tail, were obtained in a cell-free in vitro transcription system containing NTP mix, RNase inhibitor (Transgene, China), and SP64 RNA polymerase (NEB, USA). DNA templates were digested by RNase-free DNase, and then RNA transcripts were purified using phenol chloroform.
6
Synthesis of Viral Protein and Plant RNA Silencing Effectors
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Fragments of TBSV P19 (GenBank: AJ288942.1) and the derivative (R75G R78G), TCV P38 (GenBank: HQ589261.1) and a derivative (W26A W274A), CMV 2b (strain Y, GenBank: D12538.1) or 2b (strain Q, GenBank: Z21863.1), PVY Hc-Pro (GenBank: AFR11765.1), and CVYV P1b (GenBank: DQ496114.1) were amplified by PCR. Fragments of Cardamine chlorotic fleck virus (CCFV) P38 (NC_001600.1), Pelargonium flower break virus (PFBV) P38 (GenBank: DQ443018.1) were synthesized by GeneArt Strings (Thermo Fisher Scientific). DNA fragments encoding HYL1, DRB4, RDR1, RDR2, or RDR6 were amplified by PCR using cDNA prepared from RNA of A. thaliana Col-0. All the fragments were cloned into pSP64-poly(A) vector (Promega) using appropriate restriction sites. Substitution of nucleotides or addition of epitope sequences on genes was performed by PCR using plasmid clones as templates and primers containing relevant sequences. Oligonucleotides are listed in Supplemental Table S1 . Each messenger RNA was prepared from the linearized plasmids using AmpliCap SP6 High Yield Message Maker Kit (Cellscript).
7
Tox Gene Expression in Wheat Germ
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The Tox gene was cloned into pSP64 Poly(A) Vector (Promega, Madison, WI), and protein produced using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega, Madison, WI) according to manufacturer instructions.
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