Week 4: Go With The Flow

After a very eventful three day weekend filled with fun at the beach and Portland waterfront fireworks it was back to work.

On Tuesday we went sampling again to Hammond, Chinook, Willapa, Ilwaco and Rogue. We filtered the DNA onto sterivex filters and will be extracting the DNA from these sites most likely sometime next week.

We got the sequences back from last week and when I compared the sequences using BLAST on the NCBI data base we quickly discovered that the Katablepharis primers that we were using were not as specific as we had thought and therefore we will be designing new primers perhaps using the ITS or 28S regions on the rRNA gene.

The primers for the general ciliate primer on the other hand did work well. Unfortunately the product of the ciliate and general eukaryote PCR is too large for quantitative PCR. So we tested primers for ciliates and general eukaryotes that produce a smaller PCR product. Some of the primers worked, so we prepped them for cloning but unfortunately we are having some problems with the transformation. We might try again with a new vector or new cells.

I also spent part of the week putting together a presentation for Monday’s intern midterm presentations.

This week has been filled with a lot of trial and error, sometimes you just have to go with the flow.