Screening for Archael DNA: PCR, Probing, and Chemiluminescence

This week involved a whole lot of looking for Archael DNA in the E.coli clone colonies. Based off a previous experiment that used chemiluminescence to detect for the Archael 16S rRNA, we found colonies that were positive for the gene (the black spots pictured below).

Chemiluminescence is a procedure that uses a DNA probe to find target DNA. A probe is a sequence that is designed to bind to complimentary DNA. When the probe finds the target DNA and the correct reagents are added, the bound DNA and probes give off light. Using a dark room, we can then develop film showing where the probe bound to DNA, therefore telling us what colonies contain the DNA we are looking for.

            Matching the spots up with the colonies they were blotted from, we did colony based PCR on a select amount of colonies that were positive. This PCR was intended to ensure we did not have false positives and that the 16S gene was present. As is common with the chemiluminescence, there tends to be a high rate of false positives and the PCR results only showed that a few of our select colonies contained the 16S gene. Based off the PCR results, I streaked out plates of the old colonies (the ones that were positive) so they could grow on fresh plates. All of this was done on Tuesday and Wednesday.

            On Thursday, my mentor and I ran another chemiluminescence experiment on the same plates we tested for the 16S. This time we were looking for the Archael amoA gene, or the gene involved in ammonia oxidation. The 15 minute exposure did not turn up with any results. We ran a 1 hour exposure and still found no results. This may have been due to an experimental error in the preparation before the probe was introduced.

            My senior scientist wants to know if the fosmids we are working with contain a large enough insert of Archael DNA. To test these fosmids, my mentor and I our going to cut out the inserts using restriction enzymes. Our first trial started today using the enzyme Not1. We are hoping this enzyme will correctly cut the inserts out of the fosmid DNA I extracted back on June 16^th. The results will be analyzed on Monday after running PCR and a gel.

            This week I have also been preparing for a mid-term presentation that will occur this Monday. I am slightly nervous but I feel ready. I will be giving a short 5 minute presentation on what I have done and what our goals are for the rest of the summer. I hope it goes well and I look forward to learning about the other intern’s projects.