Our strain 37 was uniformly shaped ovals clumped together into groups of 3 to 4. We took photos of this organism under the light microscope. Our strain 40 showed possible signs of motility based on the twitching motion observed. Strain 40 varied between elongated ovals to round in shape, occurring in pairs. We took photos of this organism under the light microscope as well. We observed no DNA on both of our slides; probably do to the cells needing to be treated to allow the DAPI in.
An example of how the Dapi works was given by gathering biofilm on a slide. Under UV, lots of DNA could be seen with the Light Microscope.
The professor of the lab prepared our slides for the TEM. On the first go-round neither of our cells appeared, but we she changed the order of making the TEM slides, our organisms appeared. It was possible she didn’t get any cells on the copper/carbon plate. Our strain 37 occurred in clumps and appered as lumpy ovals whose membranes lacked rigidity. Our strain 40 appeared as elongated ovals with singular flagella. Images of both strains were taken
The next class we made a gel to run our PCR products in. We used size marker 3 and we were looking for fragments of about 550 basepairs. We had positive results for strains 34, 44 and 45. Possible reasons why no bands appeared for strains 27, 29 and 48 could be no DNA was extracted to begin with or an error occurred in the PCR. We also had positive results for LRH-4, 9, 11, 12 and 14. . Possible reasons why no bands appeared for LRH-1, 2, 5, and 13 could be no DNA was extracted to begin with, the samples were not the correct organism (we used specific primers), or an error occurred in the PCR. No contamination of the PCR Master Mix was indicated by the lack of bands in the DNA (-) lane. We recorded a photo of our PCR.
i believe we didn't see any cells the first time because of the way she orginally prepared them. At first she put the cells on top of the collodian, then since she couldn't see anything, she submerged them.
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ReplyDeleteDoes the DAPI undergo a conformation change? I thought it just intercalated between base pairs and then fluoresced.
ReplyDeleteTo samsung: I thought we only used collodion slides with the biofilm that she provided us with. I didn't think we put any of our samples on the collodion slides...did we?
ReplyDeleteyeah im pretty sure ms. cindy prepared our strains and the biofilm on collodion slides if thats what the carbon covered copper grid was
ReplyDeletecollodian was only for the biofilm slides. To prepare our copper grids for TEM she floated them first on a suspension of our isolates then added a drop of uranyl acetate the carbon layer is not the same as collodian
ReplyDeleteIs a negative stain actually used in TEM or does it just look the same as if a negative stain were used?
ReplyDeleteI'm pretty sure that DAPi doesn't go under a conformation change. It just fits in between the base pairs.
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