Pgem easy vector system 2

With the Dynabeads mRNA DIRECT kit (Biotech, UK), mRNA was isolated using 5 mg of freeze-dried skin secretions. A BD SMART RACE cDNA Amplification Kit (BD Clontech, UK) was used to generate full-length cDNA through reverse transcription reaction. The resultant cDNA was subjected to 3′-RACE PCR using a nested universal primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and a sense primer (S1: 5′-GGCTTYCCTGAAGAAATCTC-3′) with same program as before (36 (link)). The PCR products were purified using a Cycle Pure Kit (Omega Bio-Tek, USA) and then cloned with pGEM Easy Vector System II (Promega, USA). Products were purified and expanded to prepare for the sequencing reaction, which was completed using BigDyeTerminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequencing results were analyzed by Chromas software (version 2.6.4) and converted into amino acid sequences by the Expert Protein Analysis System (ExPASy). The open reading frame of peptide was compared with the NCBI-BLAST platform database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and multiple sequence alignments of similar peptide regions were performed through Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

With the Dynabeads mRNA DIRECT kit (Biotech, UK), mRNA was isolated using 5 mg of freeze-dried skin secretions. A BD SMART RACE cDNA Amplification Kit (BD Clontech, UK) was used to generate full-length cDNA through reverse transcription reaction. The resultant cDNA was subjected to 3′-RACE PCR using a nested universal primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and a sense primer (S1: 5′-GGCTTYCCTGAAGAAATCTC-3′) with same program as before (36 (link)). The PCR products were purified using a Cycle Pure Kit (Omega Bio-Tek, USA) and then cloned with pGEM Easy Vector System II (Promega, USA). Products were purified and expanded to prepare for the sequencing reaction, which was completed using BigDyeTerminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequencing results were analyzed by Chromas software (version 2.6.4) and converted into amino acid sequences by the Expert Protein Analysis System (ExPASy). The open reading frame of peptide was compared with the NCBI-BLAST platform database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and multiple sequence alignments of similar peptide regions were performed through Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).