Linear Plasmids, Restriction Enzyme Ligation and Plasmid Extractions

 

Linear Plasmids, Restriction Enzyme Ligation, and Plasmid Extractions

 

The project I’m currently working on is linear plasmids. We’re trying to take a plasmid from E.coli that contains a gene called tet that codes for tetracycline (an antibiotic) resistance, and remove D.rad’s (Deinococcus Radiodurans) lux.s gene before incorporating tet into the spot in D.rads genes where lux used to be.

 

On Monday we decided to separate into two groups and try two different methods of removing the lux and replacing it with tet. While the other group will be doing the original overlap PCR, our group will be trying restriction enzyme ligation. Both groups will be performing the same basics, but going about the process in different ways. In the end we’re just trying to find which process is more reliable and efficient.

 

Before we could start doing restriction enzyme ligation, we needed to create more plasmids, as the various experiments over the week have depleted our supply. Using New England Biolabs’ Monarch Plasmid Miniprep Kit, we took our original sample of E.coli and worked to get more of the plasmid containing tet.

 

 

Plasmid Extraction Protocol:

 

To start we put 1 ml of E.coli culture in the centrifuge (at 16,000 x g ~ 13,000 RPM) in order to pellet it, before discarding the supernatant.

We then resuspended the pellet in 200 μl Plasmid Resuspension Buffer, then vortexed it until there were no visible clumps of cells. At this point the liquid was a light pink.

After we lysed the cells by adding 200 μl Plasmid Lysis Buffer and inverted the tube until the liquid was a dark pink, we then let it incubate at room temperature for one minute.

Then we neutralized the lysate by adding 400 μl of the Plasmid Neutralization Buffer and inverted the tube until the liquid was yellow. We then let it incubate for two minutes, again at room temperature.

After that we centrifuged the solution for a full five minutes (again at 16,000 x g)

We then transferred the supernatant to a spin column and centrifuged it for one minute before discarding all flow-through.

We re-inserted the column into a collection tube and added 200 μl of Plasmid Wash Buffer 1, that removes RNA, endotoxin, and proteins. We once again centrifuged for a full minute and discarded flow-through.

After we added 400 μl of Plasmid Wash Buffer 2 and centrifuged the tube for one minute.

We carefully transferred the column to a 1.5 ml microfuge tube making sure not to touch any of the flow-through.

To finish we added 30 μl of DNA Elution Buffer, warmed to 50 degrees Celsius, to the center of the matrix. After waiting for a minute we once again centrifuged for a minute to elute the DNA.

 

 

After fully completing the process we nanodroped our end product to measure how successful our plasmid extraction was. We ended up getting  12.3  1.61  .89 , so we measured our original sample to see why the extraction was so unsuccessful.  The original measured at   .85  .10  600  .85  on the nanodrop. We re-ran the plasmid extraction in order to get more successful results. However this time repeated the first step five times, getting a pellet five times the size of the first.  The second plasmid extraction was much more successful. Once measured on the nanodrop it measured at   30.6  1.88  1.77  . While not as great as hoped, the second extraction was much better than the first.

 

References-

Biolabs, N. E. (n.d.). Monarch® Plasmid Miniprep Kit | NEB. Www.neb.com. Retrieved September 16, 2022, from https://www.neb.com/products/t1010-monarch-plasmid-miniprep-kit#Product%20Information

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