Biology 475 Molecular Biology Lab Three – Library Screening with Macro-Arrays and Probes Shanna Briggs Copyright 2003

Introduction  The goal of this lab was to further analyze the isolated plasmid DNA through hybridization with DIG marked probes specific for 16S of all bacteria, red bacteria and green bacteria. DIG is a plant compound not found in any other organism. These probes bind the DNA and are localized by antibodies attached to alkaline phosphotase. The blots for this procedure were made during week 2 using nitrocellulose and a vacuum pump. The hybridization was performed for us between labs periods.

	Slot-Blotting Methods
Methods: Using a 96 well plate, each student added 60 ul of each mini-prep samples, and15 ul control DNA (Roseiflexus and Chloroflexus) in the bottom row. We then divided the plate into three identical plates.
Methods: Using a multi-channel pipette, samples from each of the clones from every person in the class were added to the nitrocellulose paper, as well as red, green, and all bacteria. We made three identical blots for use later with different probes.

	Probe Hybridization Methods and Results
Methods: At the start of week 3 we washed the blot in wash buffer consisting of SSC and SDS. This wash was repeated twice at 45C and once at room temperature.
Results: This blot was hybridized with probe specific for all bacteria. It should have all the samples lit up. However, during the boiling step of the procedure some water leaked in and perhaps this affected our results. (Note: When this image is printed there are no dark spots visible)	81, 82, 83, 84, 85, 86, 87, 88, 89, 90 All Bacteria

Results: This blot was hybridized with probe specific for green bacteria. For my samples it appears that clones 82, 83, 86, and 89 may have green bacterial inserts. Further analysis of these inserts or simply repeating this procedure may prove useful.	81, 82, 83, 84, 85, 86, 87, 88, 89, 90 Green
Results: This blot was hybridized with probe specific for red bacteria. Of my samples it appears that clones 81, 82, 84, 85, 88 and 90 gave positive results (and thus are red bacteria). Two of these clones will be chosen for further analysis because the bacteria of interest are closely related to red bacteria (Roseiflexus).	81, 82, 83, 84, 85, 86, 87, 88, 89, 90 Red

Discussion

Between week 2 and 3, the instructors used the nitrocellulose papers generated during week 2 and hybridized them with probes marked with DIG specific for all bacterial 16S, red 16S and green 16S. During week 3, we washed the blots, and then applied antibodies raised against DIG. These antibodies have the enzyme alkaline phosphotase, which reacts with a horseradish substrate to produce a purple color change. This allows visualization of the DNA only at the sequences that matched the probes.

Once we receive results of our hybridization, we will be able to identify which clones had taken up the plasmids with vectors. These results will be cross checked with the results from lab two, and clones for further analysis will be chosen. The further analysis will assist in the assay of the bacterial diversity from the Yellowstone samples.

Addendum:

Now that results are available (see above) we have chosen clones 81 and 82 for further analysis because they show definite hybridization with the red probes. It is puzzling that they did not hybridize with the probe specific for all bacteria. However, since none of the samples had the results expected on that blot, we will assume that there was an error on this blot, probably caused by the water leak during the boiling step. In addition, both of these clones show definite bands for the vector and the insert on the gels from Lab #2.