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2/12-2/16: Primer Validations

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Introduction:    Primer validation is important to ensure that the primers we will be working with in our qPCR experiment with  D. aquaticus  are functioning correctly. Primer validation must be done before performing our test to see what is considered cold shock or heat shock. The plan is to validate 6 different primers and perform qPCR on them to see if the results are accurate. For each primer, 5 different dilutions are done, 1:1, 1:5, 1:25, 1:125, and 1:625. With these 5 dilutions, we are able to compare the 5 graph lines on qPCR to see if they line up, and if this is the case, there will be a efficiency value between 90-110%.   Methods:   Nanodrop values were taken on our RNA extraction products, the values are shown in figure 2.    The protocol for our RT qPCR experiment is shown below.    Figure 1: Luna 1 step RT qPCR kit protocol.    Results from the qPCR were finalized 2/12 and the data was validated in excel to d...
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Week 21 (03/20/23 - 03/24/23): Discoveries in Optimal Growth Conditions with Deinococcus Species

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Introduction:    The goal for this week was to figure out the best ways to grow each bacteria. During the last few weeks, we have had difficulties with multiple species of  Deinococcus . Each species would grow in TGY broth, however, the OD600 values were too low for the species to be used in any testing. This leads us to trying different growth methods to see if there are better ways to obtain a higher yield.    Methods:   400 ul of TGY was prepared and autoclaved. 200 ul of TGY was prepared, separated into 4 125 ul flasks, and then autoclaved. TGY with agar was heated over a hot plate and poured over plates, which were left out overnight to solidify. Media was   Gram staining was done on  D. indicus  and  D. grandis . OD600 values were taken on the nanodrop on  D. roseus ,  D. caeni ,  D. aquatilis ,  D. gobiensis , and  D. indicus . An inoculation was also performed with  D.  caeni onto a TGY agar pl...
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Week 20 (03/06/23 - 03/10/23): New Plan For Working With D. grandis

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Introduction:    Because our  D. grandis  results from 2 weeks ago showed that this bacterium was not resistant at antibiotic concentrations between 1.56 ug/ml and 100 ug/ml for all 3 antibiotics, we are shifting our focus to trying to classify the MIC for the 3 antibiotics we are testing on with an additional 3 antibiotics we have not worked on yet. The goal is to see if  D. grandis  has a higher MIC for all the antibiotics on the board in comparison to the other  Deinococcus  species.   Methods:   400 ml of TGY was prepared following the 3:3:1 method and was autoclaved overnight.   Gram staining was performed on 3/8 on select species shown in figure 3, and OD values were taken on 6 different species of  Deinococcus  on 3/10.   18 test tubes were filled with TGY and autoclaved for 30 minutes. The next day, TGY was removed from each tube to equate to the amount listed next to each test tube in red and purple ink in fi...
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Week 19 (02/27/23 - 03/03/23): Continuation of MIC/Antibiotic Testing

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  Introduction:  The goal for this week is to complete our protocol for the MIC testing with  D. aquaticus  and start a new procedure with  D. roseus  to identify if it will respond to methicillin, kanamycin, or ofloxacin. We are repeating the same procedures we have done in the previous weeks with both of our tests performed. The main outcome we are hoping for is to identify the MIC for  D. aquaticus,  and to see what antibiotics work with  D. roseus  to see if there are similarities with the previous  Deinococcus  species that underwent antibiotic testing:  D. aquaticus  and  D. grandis .   Methods:    MIC testing with D. aquaticus: 8 test tubes containing TGY with 0.5% agar were autoclaved and set out to cool. The 8 test tubes consisted of: 1 negative control tube, 1 positive control tube with no antibiotic added, 2 3000 ng/ml tubes, 2 2000 ng/ul tubes, and 2 1000 ng/ul tubes. The 2 mg/ml stock ...
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