Journal: Biological Procedures Online
Article Title: Analyzing ligation mixtures using a PCR based method
Figure Lengend Snippet: PCR products from ligation mixtures using vector specific primers M13-R M-13F. Electrophoresis on 1.2 % agarose gel and staining with Ethidium Bromide. A: Band 1 derived from recombinants and Band 2 from relegated pCR 2.1 vector. Lane 2 3 has two types of recombinant specific products and both were used for calculation band intensity/amount. B: PCR product amounts calculated as corresponding band intensity (mean grey value) using ImageJ 1.33 software (http://rsb.info.nih.gov/ij) .
Article Snippet: Reagents Plasmid cloning vector: pCR 2.1 plasmid (Invitrogen, Carlsbad, CA, USA) Restriction enzymes: EcoR I, Hind III DNA ligase: T4 DNA ligase Competent cells: One Shot® Top10 E.coli chemically competent cells (Invitrogen, Carlsbad, CA, USA) Milli-Q water Plasmid specific primers: M13-forward and M13-reverse primers PCR reagents: 10X reaction buffer, dNTP mix (2.5 mM each) and Taq DNA polymerase Eppendorf/Brinkmann, Westbury, NY, USA) Agarose: SeaKem LE agarose (BioWhittaker Molecular Applications, Rockland, ME, USA) Ethidium Bromide.
Techniques: Polymerase Chain Reaction, Ligation, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Derivative Assay, Recombinant, Software