cDNA cloning and the sequencing were conducted for medaka ccni because the cDNA sequence was not available from public databases. It was determined by PCR using a KOD FX Neo-DNA polymerase (Toyobo, Osaka, Japan) and medaka ovary cDNA. The primers used were Cyclin I 5′-SS and Cyclin I 3′-AS (Table S1 ). The amplified products were phosphorylated, gel-purified, inserted into the pBluescript II vector (Agilent Technologies, Santa Clara, CA, USA), and sequenced. The sequence determined was deposited into the DDBJ/GenBank/NCBI database (accession number LC435346).
Pbluescript 2 vector
Manufactured by Agilent Technologies
Sourced in United States
The PBluescript II vector is a high-copy number plasmid commonly used in molecular biology for cloning and expression of DNA sequences. It features a multiple cloning site, a lac promoter, and an ampicillin resistance gene.
Automatically generated - may contain errors
Lab products found in correlation
Kod fx neo dna polymerase, by Toyobo (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Pegfp c1 vector, by Takara Bio (1 mentions)
Pmcherry c1 vector, by Takara Bio (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Omniscript reverse transcriptase, by Qiagen (1 mentions)
3 protocols using pbluescript 2 vector
1
Medaka ccni cDNA Cloning and Sequencing
2
Generating Isogenic FUS-P525L Mutant hiPSC Line
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For generating an isogenic FUSP525L/+ mutant line, the control hiPSC line 201B7 was cultured under feeder-free conditions using StemFit AK03 as the manufactured protocol. Human codon optimized spCas9 cDNA was sub-cloned into pCAGGS vector [8 (link)], and human U6 promoter, CRISPR target sequence and sgRNA scaffold sequence were sub-cloned into pBlueScript II vector (Agilent Technologies). As donor DNA, 90 bp of FUSP525L phosphorothioate modified single stranded OligoDNA (PS-ssODN) and FUSWT PS-ssODN with silent mutations were synthesized. pCAGGS-spCas9 vector, pBlueScript II sgRNA expression vector and PS-ssODN were co-electroporated with Puromycin N-acetyltransferase expression plasmid using Neon Transfection System (Thermo Fisher Schientific). After puromycin-resistant single hiPSCs colonies were picked up, PCR genotyping and Sanger sequencing were performed to identify the knock-in clone. Thus, the FUS-mutant isogenic line (FUSP525L/+) were established. The FUSP525L/+ line was validated for pluripotency markers, normal karyotypes, genomic integrities, and developmental potency (data not shown).
3
Cloning and Expression of Key Signaling Proteins
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Mouse Braf, Map2k1 (MEK1), and Smad2 cDNAs containing the entire coding regions were cloned from mouse brain mRNA by reverse transcription (RT)-PCR using Omniscript Reverse Transcriptase (Qiagen) and Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific). The primer sets used are indicated in S1 Table .
These cDNAs were digested with XhoI, BamHI, and EcoRI, respectively, and subcloned in pBluescript II vector (Agilent Technologies). Constitutively active B-Raf(V637E) and MEK1(S218D/S222D) cDNAs were constructed by introducing point mutations into wild-type cDNAs in the vector by PCR. The primer sets used are indicated inS2 Table .
These PCR products were self-ligated. Braf and Map2k1 cDNA fragments excised with XhoI and BamHI, respectively, were inserted into pEGFP-C1 vector (Clontech) in frame with the EGFP-tag. Smad2 cDNA fragment excised with EcoRI was inserted into pmCherry-C1 vector (Clontech) in frame with the mCherry-tag. pEF-BOS/Myc-H-Ras(G12V) was constructed as described previously [28 (link)].
The recombinant plasmids were transfected to RLE cells with Lipofectamine LTX mixed with Plus Reagent (Life Technologies).
These cDNAs were digested with XhoI, BamHI, and EcoRI, respectively, and subcloned in pBluescript II vector (Agilent Technologies). Constitutively active B-Raf(V637E) and MEK1(S218D/S222D) cDNAs were constructed by introducing point mutations into wild-type cDNAs in the vector by PCR. The primer sets used are indicated in
These PCR products were self-ligated. Braf and Map2k1 cDNA fragments excised with XhoI and BamHI, respectively, were inserted into pEGFP-C1 vector (Clontech) in frame with the EGFP-tag. Smad2 cDNA fragment excised with EcoRI was inserted into pmCherry-C1 vector (Clontech) in frame with the mCherry-tag. pEF-BOS/Myc-H-Ras(G12V) was constructed as described previously [28 (link)].
The recombinant plasmids were transfected to RLE cells with Lipofectamine LTX mixed with Plus Reagent (Life Technologies).
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