This week began with Dr. Rainey soaking up the rays in Mexico while we were trapped in a dark basement looking at slides J JK! We very much enjoyed our field trip to the basement to do some microscopy. We started off by suspending some cells from our selected strains in .5ml H20 using a wooden stick (unsterilized, eww). We then pipetted 1.5µl of the liquid onto a coverslip and placed the slide onto the coverslip. We were explained to that you don’t want to have more than 2µl of liquid on the coverslip or everything will just be swimming around and hard to focus and take pictures of under the microscope. We also stained the slides with DAPI, 4'-6-Diamidino-2-phenylindole, which forms fluorescent complexes with natural double-stranded DNA. When viewed under UV light you are able to see the organisms’ DNA. Unfortunately we did not have much luck with this stain. We believe this is due to the hydrophobic and membrane characteristics of these organisms. We did however view our slides under the light microscope using differential interference and were able to see our organisms. Strain 17, AT03-34-2, from our group appeared to be little round cells in groups of two that were trying to get to the edge of the water/air line. (sorry cant get image to insert properly but its image 1)
We also attempted to view our organisms using the TEM or transition electron microscope. TEMs use electrons as a light source and have a much lower wavelength making it possible to get a resolution significantly better than with a light microscope. We prepared a copper grid by dipping it into our cell/h20 mixture and then into uranyl acetate. Liquid nitrogen was added to the TEM to condense the molecules. The screen we viewed the slides on gives off visible light by electron excitation. For most of the slides we were not able to see anything. She decided to set up the slides differently by collecting the water from the bottom of the drop rather than the top in hopes of being able to see more. Strain 40 ended up having a cell with a very long, defined flagellum. We were also able to view strain 17 again and saw the same pairing of cells. Overall it was interesting and a fun day! I also highly enjoyed myself at the Hornets game that night!!
Wednesday morning I began the process of extracting DNA using the MOBIO bead beating kit for strains that we do not have 16S information for. In class that afternoon we ran our PCR from last week on gel and recorded data for our temperature and cellulose plates. To the xylan plates we added iodine and to the avicell and granular cellulose plates we added congo red let it sit for fifteen minutes then added NaCl. Unfortunately the results for all strains all tests was negative L Our PCR was semi successful. The first six were strains from our collection and were all positive. The rest were our N97 soil dilution colonies. Only two, N97-6 and N97-19 were weakly positive and suspected Geo’s due to the presence of a band at about 564bp. We will use 16S primers for these. In the image below you can see our gel. The first six are from the strain collection and row one column 11 is N97-6 and row 2 column 9 is N97-19.
We also attempted to view our organisms using the TEM or transition electron microscope. TEMs use electrons as a light source and have a much lower wavelength making it possible to get a resolution significantly better than with a light microscope. We prepared a copper grid by dipping it into our cell/h20 mixture and then into uranyl acetate. Liquid nitrogen was added to the TEM to condense the molecules. The screen we viewed the slides on gives off visible light by electron excitation. For most of the slides we were not able to see anything. She decided to set up the slides differently by collecting the water from the bottom of the drop rather than the top in hopes of being able to see more. Strain 40 ended up having a cell with a very long, defined flagellum. We were also able to view strain 17 again and saw the same pairing of cells. Overall it was interesting and a fun day! I also highly enjoyed myself at the Hornets game that night!!
Wednesday morning I began the process of extracting DNA using the MOBIO bead beating kit for strains that we do not have 16S information for. In class that afternoon we ran our PCR from last week on gel and recorded data for our temperature and cellulose plates. To the xylan plates we added iodine and to the avicell and granular cellulose plates we added congo red let it sit for fifteen minutes then added NaCl. Unfortunately the results for all strains all tests was negative L Our PCR was semi successful. The first six were strains from our collection and were all positive. The rest were our N97 soil dilution colonies. Only two, N97-6 and N97-19 were weakly positive and suspected Geo’s due to the presence of a band at about 564bp. We will use 16S primers for these. In the image below you can see our gel. The first six are from the strain collection and row one column 11 is N97-6 and row 2 column 9 is N97-19.
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