Abi prism 3100 avant

DNA oligonucleotides were purchased from Sigma-Aldrich (Ishikari, Japan), and stored as 100 mM stock solutions in 10 mM Tris-HCl with 1 mM EDTA (pH 8.0). WT human genomic DNA was purchased from Promega (Tokyo, Japan). Heterozygote human genomic DNA with seven mutations (G12A, G12C, G12D, G12R, G12S, G12V and G13D) in the KRAS gene codon 12 and 13 were obtained from Horizon Diagnostics (Cambridge, UK). Eprobes (shown in Table I) were obtained from K.K. DNAFORM (Yokohama, Japan). Point mutations in codon 12 of the KRAS gene (GAT, GCT, GTT, AGT, TGT) were cloned into the plasmid pGEM-T (Promega) as previously described (26 (link)) and stored in glycerol at −80°C for long-term conservation. Point mutation of codon 13 (GAC) in the KRAS gene was prepared using the same protocol. The KRAS codon 12 (CGT) point mutation was prepared using the synthetic DNA ordered and inserted into a pUC57 plasmid (GenScript, Tokyo, Japan). The sequences of WT and all seven mutant clones were verified on ABI PRISM 3100 Avant (Applied Biosystems, Tokyo, Japan), diluted in TE buffer and stored at −20°C until use.

Viral RNA was isolated from the allantoic fluid of infected chicken embryos with a commercial QIAamp Viral RNA mini kit (Qiagen, # 52904). Full-length viral genome segments were obtained by reverse transcription and PCR with specific terminal primers, MMLV, and Taq-polymerase (Alpha-Ferment Ltd., Moscow, Russia). The amplified fragments were separated by electrophoresis in 1–1.3% agarose gel and subsequently extracted from the gel with the Diatom DNA Elution kit (Isogene Laboratory Ltd., Moscow, Russia, # D1031). Sequencing reactions were performed with terminal or internal primers with the BrightDye™ Terminator Cycle Sequencing Kit v3.1 (Nijmegen, The Netherlands), followed by analysis on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems 3100-Avant Genetic Analyzer, Foster City, CA, USA). The Lasergene software package (DNASTAR Inc., Madison, WI, USA) was used for the assembly and analysis of nucleotide sequences.

Genomic DNA from Yucatan fibroblasts was extracted and 40 ng used as template in PCR reactions using Pfu polymerase (Promega, Madison, WI, USA) with primers listed in table S1.
Amplified PCR products were sequenced on the ABI Prism 3100-Avant (Applied Biosystems, Foster City, CA). The partially complete Yacatan minipig Smn1 sequence was submitted to GenBank database under accession number KF585502.

Viral RNA was isolated from the allantoic fluid of infected chicken embryos with a commercial QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany, # 52904). Full-length viral genome segments were obtained by reverse transcription and PCR with specific terminal primers, MMLV, and Taq-polymerase (Alpha-Ferment Ltd., Moscow, Russia). The amplified fragments were separated by electrophoresis in 1–1.3% agarose gel and subsequently extracted from the gel with the Diatom DNA Elution kit (Isogene Laboratory Ltd., Moscow, Russia, # D1031). Sequencing reactions were performed with terminal or internal primers with the BrightDye ™ Terminator Cycle Sequencing Kit v3.1 (Nimagen, Nijmegen, The Netherlands), followed by analysis on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems 3100-Avant Genetic Analyzer, Foster City, CA, USA). The Lasergene software package (DNASTAR Inc., Madison, WI, USA) was used for assembly and analysis of nucleotide sequences.

M-Sec cDNA (GenBank accession No. NM_009396.1) was generated from mouse splenic macrophage mRNA by reverse transcriptase polymerase chain reaction and inserted into pBluescript II KS (+) (Agilent Technologies) as described previously10 (link). The BamHI-SalI fragment was subcloned into the mammalian expression vector pEGFP-C1 (Clontech Laboratories, Inc.), pStrawberry-C110 (link) or a bacterial expression vector pGEX4T-2 or pGEX6P-1 (GE Healthcare) to produce recombinant glutathione S-transferase (GST)-fusion proteins. Deletion and point mutants of M-Sec were generated using KOD-Plus-Mutagenesis kit (Toyobo Life Science) following the manufacturer’s instruction. The primers used for the mutagenesis are listed in Supplementary Table S1. Products were sequenced using an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems). We have optimized transfection conditions for the individual plasmid constructs by checking protein expression levels with Western blotting analysis (Supplemental Fig. S6). The plasmids encoding PLCδ-PH-GFP and Akt-PH-GFP were obtained from Addgene (plasmid No. 21179 and 18836, respectively).

The obtained PCR products were checked on an agarose gel and then enzymatically purified with the EXO-SAP mix (Jena Bioscience, Jena, Germany) according to the manufacturer’s protocol. Sequencing reactions consisted of 2.5 µL of clean PCR product, 5 µL of H2O, and 2.5 µL of primer (10 µM). Cycle sequencing was performed by GATC Biotech (Konstanz, Germany) with the BigDye Terminator cycle sequencing kit on an ABI Prism 3100 Avant automated sequencer (Applied Biosystems, Foster City, CA, USA).

Genomic DNA was digested with a two enzymes combination, EcoRI and MseI [71 (link)]. Then EcoRI and MseI adapters were ligated using an AFLP Core Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. A total of 12 AFLP primer combinations, generated by four EcoRI primers (EcoAGC, EcoACT, EcoACA, EcoACG) combined with three MseI primers (MseCAA, MseCTA, MseCAC) were used. A pre-amplification PCR reaction was executed with primers based on the adapter sequences with one additional selective nucleotide at the 3’ end of each primer (EcoA + MseC). Using an aliquot of the PCR product, a selective amplification reaction was performed with Eco and Mse primers combination with three additional selective nucleotides at the 3’ end of each primer. Each 5’ end of the EcoRI primers was labelled with different fluorescent dyes (PET, FAM, NED, and VIC). AFLP fragment analysis was performed on an automatic capillary eletrophoresis sequencer ABI PRISM 3100-Avant (Applied Biosystems, Foster City, CA). The data were analysed using GeneScan (Applied Biosystems) and Genotyper DNA Fragment Analysis (Applied Biosytems) software as above. Only AFLP bands that were present in in S. incanum and absent in S. melongena parental lines were scored.