Microscopy and qPCR

    This week we learned about microscopy as well as did RNA isolation, qPCR and inoculated a number of biochemical tests. 

    Chad spent one day teaching us about the florescent microscope and showing us the basic parts and procedures. We learned about the four channels that showed different colors and different parts of cells, like blue for the nucleus and green for the cytoskeleton/cell wall and red for cytosol. He showed us how to prepare the slide then went through the procedure, and showed us around the program. 

    This week we did the RNA isolation again. The UV exposure was performed at 70 mJ/cm^2 for two minutes, with the E.coli being grown that morning then normalized, exposed and diluted. After the 30 minute recovery at 30°C we moved on to RNA isolation. With one round of bead beating we did the procedure like normal with one change. Instead of working out of a normal tube holder i switched to working out of an ice box after the bead beating step and extraction was over. The procedure went much better and although we did end up doing an ethanol precipitate after we were able to do qPCR the next day. 

Post RNA isolation

T1

31.2

2.02

1.59

T2

47.5

1.73

0.89

C1

70.2

2.03

1.42

C2

63.1

2.00

1.65

Post Ethanol Precipitation

T1

42.0

2.02

1.77

T2

24.7

2.11

1.28

C1

25.1

2.11

1.29

C2

21.7

1.94

1.61


    We used T1 and C1 for the qPCR and calculated what needed to go into each tube and each well. 

Tube 1 (T1)
- 16μl of T1 sample
- 13.6 water

Tube 2 (C1)
-8μl of C1 sample
-21.6μl water

Tube 3 (master mix)
-80μl reaction mix
-8μl enzyme mis
-6.4μl LexA Forward
-6.4μl LexA Reverse

Tube 4 (control)
-36.8μl water
-1.6μl LexA Forward
-1.6μl LexA Reverse


Wells A1 A2 and A3 each got 7.4μl of tube 1 and 12.6μl of tube 3
Wells B1 B2 and B3 each got 7.4μl of tube 2 and 12.6μl of tube 3
Well H1 got 20μl of tube 4



    In the end the qPCR went well, however the change was not enough to be significant meaning the bacteria wasn't being stressed enough. Because of this we will have to redo this test with higher exposure to ensure that it works. 

    For the biochemical tests we had to make the citrate plates and pour the starch plates, and we were able to inoculate the Lactose, Sucrose, MRVP, starch and citrate tests as well as the various TGY and R2A plates from last week. These tests will incubate over the weekend and we will be able to see the results next week. 
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