Biology 475

Molecular Biology

Lab Three- Library Screening with

Macro-Arrays and Probes

Rogan Rattray

Copyright 2003

Introduction

Preparing a macro-array for probing our library was done by dot-blotting our samples onto nitrocellulose paper. The arrays were then screened using probes that were sensitive to sequences of 16S DNA that are found in Red or Green bacteria. A probe that should interact with 16S DNA from all bacteria was also used as a control.

	Blotting Methods
Methods: Denatured plasmid samples were placed onto the nitrocellulose paper by putting the appropriate samples into the corresponding wells of the blotting apparatus (right) and applying a vacuum to draw the samples into the paper.
Methods: The blots were rinsed several times to insure that they were free of contaminants before we applied the specific probes. (photo courtesy of Norm McIntosh)

	Probe Hybridization Methods and Results
Methods: The blots were incubated with a probe specific for 16S sequences found in Red Green and all Bacteria. The hybridized samples were then treated with enzyme linked antibodies that are specific for the protein that had been linked to the DNA probe. The final step involved developing the hybridized blots (right) with a substrate that the enzyme would convert into a precipitate that we would see in the location of hybridized sample. (photo courtesy of Norm McIntosh)
Results: The dots that are visible indicate samples where the probe hybridized with the DNA sample. This blot (right) was treated with the probe that is specific for sequences found in Red bacteria.

Discussion

The row immediately above the row with the darkest spot (bottom left of picture) was the row made from the samples that I had worked with in the previous lab. Though some of these dots are faint, it is notable that there are six dots that correspond to sample numbers that had previously been shown to have 16S plasmid inserts (see lab 2 results). This assay shows that those inserts were cloned from Red mat-sample bacteria. We now know specifically which samples to concentrate further study to, such as sequencing and computational analysis of those sequencing results.