Biology 475

Molecular Biology

Lab Three – Library Screening with

Macro-Arrays and Probes

Kelly L. Shipley

Copyright 2003

Introduction For this procedure we will be screening our already cloned DNA from our PCR amplification for the presence of an insert. We have already used restriction enzymes to cut and determine presence of an insert. Now we will use the hybridization of our sample with probes to verify and identify inserts. These probes are visually labeled and will be applied to nitrocellulose paper containing our samples.

	Slot-Blotting Methods
Methods: Using the multi-channel pipettor, we transfer our SSC to the wells of the 96 well plate through the blotting apparatus onto the nitrocellulose paper. The SSC wets the wells as the pump pulls it through to the paper.
Methods: After our samples are completely denatured we turn the valve to “Atmosphere & Blotter” setting. At this setting samples are drawn through to the nitrocellulose paper gently.

	Probe Hybridization Methods and Results
Methods: During several stages of the macroarray procedures it is necessary to rotate the nitrocellulose paper in its solution on the orbital mixer to ensure proper covering of the paper with the solution. Our macroarray blots were placed on the orbital mixer for 30 min. in blocking solution, and 30 min. in Ab solution as well as for the “wash cycles.”
Results: After completing all of the above steps, we should have color changes on our macroarrays. We do not have any results from the array however. They did not hybridize for a long enough period of time before we performed all of the above, so our results show negative for the time.

Discussion

Our restriction digested samples have shown the presence of plasmid and insert through gel electrophoresis. In this procedure we use macroarray blots on nitrocellulose paper to determine the presence of plasmid and insert.

Probe hybridization requires we take our macroarrays of samples and add probes to them specific to 16s rRNA. For this we used red-specific, green-specific and all bacteria-specific probes, one on each of the three macroarrays, hoping to find mostly red bacterial 16s rRNA. The probes are specific to DNA we are looking for. The probes are also covalently attached to DIG, a plant compound. Using this compound, we can add antibodies to the macroarray which will attach specifically to the DIG. The antibodies which are attached to an enzyme will then change color when a specific substrate is added. In this ELISA-like procedure we can determine the presence of our target DNA insert in our sample through color changes on the nitrocellulose paper.

Unfortunately at this time we do not have results to our probe hybridization. The probes are ideally left to attach to the macroarray for 12 hours and ours only were exposed to the probe for 3-4 hours. This significant decrease in exposure time to the probe was not enough. Obviously the probes did not have enough time to attach, therefore the rest of the steps did not function correctly either. If the probes did not attach, then the Ab could not attach to the probe and the substrate could not attach to the enzyme. In a chain reaction starting with the absence of probes, our macroarray was not successful.

At this time, we are exposing the macroarray with our samples to the probes again. Hopefully we will see more positive results to this next trial. We expect to see results correlating to our gel electrophoresis results showing plasmid and insert in samples 51, 52, 53, 55, 56, 63 and 66.