Biology 475

Molecular Biology

Lab Three – Library Screening with

Macro-Arrays and Probes

Andy Mikles

2003

Introduction In this lab we did western blot and probe analysis of the blot. (we actually made the blot the week before.) we made 3 duplicate blots in order to run 3 different tests. On one blot, we hybridized with a green bacteria specific probe, one with a red bacteria specific probe, and the third was just a bacteria specific probe. This probe will bind to the sequence of the specific bacteria it is made for, and by an ELISA like test we can see color changes of the bacteria present in our blots.

	Slot-Blotting Methods
Methods: We first put each of our samples into a 96 well plate and made 3 exact copies of that. We then loaded all the sample from 1 plate onto 1 blot. We repeated this procedure twice more, so we could have 3 replica plates, one for each of our 3 probes
Methods: We repeated this procedure twice more, so we could have 3 replica plates, one for each of our 3 probes

	Probe Hybridization Methods and Results
Methods: we washed our blots, added blocking solution so that the nitrocellulose wouldn’t bind any of our antibody when we add that. We then added antibody, which binded to the DIG attached to the probe. The antibody had an enzyme linked to it which would change color in our substrate. So when we finally added our substrate, there would be a color change where the probe was present.
Results: here are the results form our probe hybridization. The spots represent color changes where the probe is present. This particular blot is of the red bacteria specific probe. Therefore, any spots represent the presence of red bacteria or red bacteria like 16s genes. My lanes were across the top and the results show I have red bacteria in lanes 1, 4,and 5.

Green blot	Red blot

Discussion

In this lab, we took the vectors that were isolated form the last lab, and performed a probe hybridization test on them in order to figure out which bacteria were represented by our samples. In order to do this, we used 3 different probes. One that would recognize part of the 16s gene of green bacteria, one that would recognize the 16s gene of red bacteria, and one that would recognize the 16s gene of all bacteria in general. We put this into a solution which stood overnight in order to attach itself to the right DNA sequences (we didn’t actually do this, but Danny did.) Our probe was attached to DIG, a unique plant compound that occurs in only one type of plant. We can then use the DIG which is attached to the probe to identify where on the blots our probes are. To do this, we used an anti-DIG antibody which was attached to an alkyline phosphatase enzyme, which would bind to the probes. We could then add a horseradish peroxide substrate, which changes color when present with the enzyme. This all worked very well and the results coincided nicely with my electrophoresis of lab 2. The electophoresis showed that #’s 1,4,5,8, and 9 took up vectors, and in the blots these same samples were positive with the general bacteria probe. My blots also showed that lanes 1,4, and 5 were red bacteria, but nothing showed up for 8 and 9 in either of the red or green tests, leading the conclusion that they are bacteria, but are not red or green like.