Identifying Genomic and Plasmid DNA Sequences in E.coli by Colony PCR. (IND-21)
16S-ribosomal RNA genes which are located on the E. coli chromosome and the ampicillin-resistance gene on a plasmid were amplified by colony PCR using a single E. coli colony as template. From left to right, samples are DNA markers, the 16 S -ribosomal RNA gene,the ampicillin-resistance gene and both genes.
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Purified DNA is most often used as a template in the PCR reaction. However, it is possible to amplify specific DNA sequences without DNA purification by starting with a single living E. coli colony. This technique is known as colony PCR and provides a powerful and reliable method for the rapid amplification and isolation of any gene in the E.coli genome or any gene on a plasmid that is carried by E.coli. In this exercise, students carry out colony PCR starting with a culture of E. coli that carries an ampicillin-resistance gene on plasmid pUC18. They first streak the cells over an nutrient agar plate in order to produce single E.coli colonies. These colonies are then used directly as templates in a PCR reaction in order to amplify a segment of the 16 S -ribosomal RNA genes which is located on the E. coli chromosome and a segment of the ampicillin-resistance gene on the plasmid. Sufficient materials are provided so that 8 groups of students can perform the experiment with each group amplifying the ribosomal RNA gene, the ampicillin-resistance gene or both genes. Typical results of the experiment are shown.
A DNA Thermal Cycler, electrophoresis equipment and Electrophoresis Package 3/4 or equivalent are needed but not included.