Gels and New Projects


    I started this week with running a simple gel and extracting bands from it. We first ran a test gel to insure everything was as expected, and once that was confirmed we loaded the remainder of the sample into special double wide lanes to accommodate the increased volume. After running those gels, we cut out the DNA we wanted, and performed a get extraction. We used the gel extraction to remove any extra DNA, and isolate the specific DNA band we want. 

    We ran four samples, two of the left fragment and two of tet, aka L1, L2, T1, and T2. 




the original confirmation gel



 

 L1

L2

T1 

T2 

 original tube mass (mg)

 988.3

991.5 

990.4 

996.0 

 tube mass with gel (mg)

1323.8 

1334.1 

1247.0 

1281.5 

 gel mass (mg)

 335.5

342.6 

256.6 

285.5 

 amount of buffer needed (3x gel mass) (μl)

 1006.5

1027.8 

769.8 

856.5 



    After that we discussed me and another TRAIN student starting on an additional project. We will be trying to break open D. rad with lysozyme, and once we have confirmation it works in our lab, moving on to break open Deinococcus aquaticus. We spend a day reading through the original project paper and creating a protocol outline. 


     We start by making an overnight culture of our bacteria, and the next day transferring 1ml of that into 200 ml of TGY Media. 

     After incubating for 24-36 hours at 30°C, we harvest the cells. 

     After pelleting the cells we was twice with multibuffer (50mM sucrose, 10 mM tris-HCL, 40 mM EDTA, 0.1% triton x-100 pH 8.0)

    Then we lyse by innoculating with lysosime for 5 minutes at 37°C. 

    We nanodrop to show DNA, showing if we were successful at breaking open the cells. 

    If we want to locate specific plasmids, we isolate them using the alkaline lysis method, and run them on gel (0.9% for intact plasmids and 1.2% for digested plasmids.)


Next week we will start by creating our overnight culture, then moving through the procedures. 





Kikuchi, M., Kitayama, S., Sjarief, S. H., & Watanabe, H. (1994). Plasmids in Several Strains Deinococcus radiodurans [Review of Plasmids in Several Strains Deinococcus radiodurans]. Radiation Research, 139(1), 123–125. JSTOR. https://www.jstore.org/stable/3578752

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