m13 forward sequencing primer  (Eurofins)

Journal: Nucleic Acids Research

Article Title: RNA self-splicing by engineered hairpin ribozyme variants

doi: 10.1093/nar/gkab1239

Figure Lengend Snippet: Analysis of splice reactions. ( A ) Denaturing PAGE (8%) of the splice assays shown in Figure – . Visualization by fluorescence imaging (ATTO680 labelled 5′EX/5′HPR/3′HPR) on a LI-COR4300 DNA Sequencer. ( B ) Reaction yields corresponding to assays shown in Figure – . 5′-bar (blue): data obtained with ATTO680 labelled 5′-EX OV or 5′HPR. 3′-bar (orange): data obtained with ATTO680 labelled 3′EX or 3′IN or 3′HPR. Analyses were performed in triplicate measurement. ( C ) Sequencing analysis of the isolated splice product 5′EX-3′EX. RNA corresponding to the assigned ligated exons was isolated from a preparative splice reaction and reverse transcribed into cDNA using an extended RT primer with a binding site for a sequencing primer. To integrate another primer binding site on the opposite end, a PCR reaction was executed, generating a dsDNA composed of the 5′EX-3′EX sequence in the centre flanked by binding sites for sequencing primers. Specific (SEQplus) forward and reversed M13 sequencing primers were used to allow for analysis of the rather short DNA by Sanger sequencing.

Article Snippet: The generated dsDNA strands were mixed with SEQplus sequencing primer (M13 forward and reversed) from Generi Biotech, necessary for the sequencing of short DNA strands, and were sent to Eurofins for Sanger sequencing.

Techniques: Fluorescence, Imaging, Sequencing, Isolation, Reverse Transcription, Binding Assay