Week 4/22

Introduction
This week, the results for the UV test done on D. xinjiangensis were observed and a plasmid extraction was performed on D. sonorensis.

Procedure
The plasmid extraction was performed as follows:

 -Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube

 -Centrifuge the tube for 30 seconds at maximum speed and discard the supernatant

-Add 600 μl of TE or water to the bacterial cell pellet and completely resuspend it

-Add 100 µl of 7X Lysis Buffer to the tube and mix by inverting 4-6 times and proceed to the next step within two minutes

-Add 350 µl of cold Neutralization Buffer to the sample and mix thoroughly until the sample turns yellow

-Centrifuge the tube for 2-4 minutes at 11,000 – 16,000 x g

-Transfer the supernatant (~900 µl) to the Zymo-Spin IIN column without disturbing the cell debris pellet

-Place the column in a Collection Tube and centrifuge for 15 seconds

-Discard the flow-through and place the column back into the collection tube

-Add 200 µl of Endo-Wash Buffer to the column and centrifuge for 30 seconds

-Add 400 µl of Zyppy Wash Buffer to the column and centrifuge for 1 minute

-Transfer the column to a clean 1.5 ml microcentrifuge tube

-Add 30 µl of Elution Buffer directly to the column matrix

-Let it stand for one minute at room temperature, then centrifuge for 30 seconds to elute the plasmid DNA

Results

Unfortunately, the only growth to be seen on any of the plates was one colony of staph contamination on one of the plates. We believe the reason for this to be that the level of UV radiation was too high, and the staph contamination likely occurred when transferring the sample from recovery broth to plate.


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