Biology 475 Molecular Biology Lab Three- Library Screening with Macro-Arrays and Probes Elizabeth Jacobsmuhlen Spring 2003

Introduction This lab will complete the macro-arrays that were started week 2. The hybridization protocol was completed for us between week 2 and 3. Week three we performed the blot washing and probe detection of the macro-arrays. The goal of this lab is to determine (via a color change) the samples that red, green, and all bacteria probes adhered to. This is another way of identifying whether or not Red, Green, or no bacteria are present in our sample.

	Slot-Blotting Methods
Methods: This is a vacuum slot-blot apparatus that allows a fast addition of multiple samples onto nitrocellulose paper. The blue apparatus in the hand shown is a multi-channel pipet tip which allows transfer of multiple rows of sample onto the vacuum apparatus. The wells on the vacuum apparatus hold the sample that had buffer added to it until all samples are loaded. Then the vacuum can be turned on and samples transferred onto the nitrocellulose paper.
Methods: Picture to the right is of the nitrocellulose paper after it had samples loaded onto it via the apparatus in the above right picture. This paper will then be laid into denaturing solution and then left in the air to dry. Then the paper will be used for probe hybridization and detection methods later in this lab.

	Probe Hybridization Methods and Results
Methods: The probe hybridization protocol was taken care of for us between week 2 and 3. This involved applying the probe to the DNA samples obtained week 2. The blots then had to be washed of the probe solution, washed in Anti DIG Ab so that it would attach to the DIG probe, and then probe detection took place. If the probe was successfully attached to the DNA on the nitrocellulose paper then a color change will take place. The picture to the right shows one of many washing steps.
Results: My sample is the third row from the top. The probe used was different as labeled on the different blots as red, green, or all bacteria. Column 1: clone #71 Column 2: clone #72 Column 3: clone #73 Column 4: clone #74 Column 5: clone #75 Column 6: clone #76 Column 7: clone #77 Column 8: clone #78 Column 9: clone #79 Column10:clone #80	Red Bacteria Green Bacteria All Bacteria

Discussion

This lab uses three probes one for red, one for green, and one for all bacteria. By using all three types of probes it allows the detection of both red and green and the use of an all bacteria probe should show if the probe was successful. A comparison can be made between the all bacteria probe and the red or green probe to determine if that particular probe was successful for the type of bacteria in the samples. If nothing shows up for the all bacteria nor red nor green then it can be supported that that particular probe is unsuccessful for the type of DNA collected in the sample.

The blots shown above are inconclusive for red, green, and all bacteria. The controls were loaded onto row five and were chloroflexis (green/column one) and rosieflexis (red/column two). The red control was positive for both red and green and the blots made from the placement of the wells were difficult to read. The green control was positive as it should have been, but the blots were not readable. All bacteria control red and green control should have both been positive and were not, as well as the blots difficult to interpret.