User:Melissa Novy/Notebook/CHEM-571/2012/10/17

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Objectives

  • Prepare plasmids for gel electrophoresis and add DPN1 to digest methylated (original) DNA.
  • Centrifuge Au/BSA solutions made on 2012/10/16 for subsequent AAS analysis.

Au/BSA Solutions for AAS

  • All solutions, regardless of whether they contained fibers, were centrifuged at 2000 rpm and 25°C for 5 min.
  • After 5 min, it was observed that the fiber-containing solutions did not form pellets, while the homogenous solutions retained their purple color. The solutions were then centrifuged for an additional 10 min at the same rpm and temperature.
  • After this second centrifugation, there appeared to be no change in the solutions from the previous observations, so the solutions were centrifuged at 2500 rpm for 5 min.
  • After the third centrifugation, no changes were observed yet again, so the solutions were centrifuged at 3000 rpm for 5 min.
  • After the fourth centrifugation, no changes were observed yet again, so the solutions were centrifuged at 4700 rpm for 15 min.
  • The test tubes containing 60 and 140 Au/BSA solutions broke in the centrifuge, so these solutions will have to be remade at a later date.
  • As the fibers in some solutions had not yet formed a pellet, these solutions were pipetted from their test tubes into Falcon tubes, leaving the fibers behind in the original container.


  • The AAS was calibrated with eight Au standards at concentrations of 5, 8, 10, 15, 20, 25, 30, and 40 ppm, made by Dr. Miller on 2012/10/08 and blanked with 1 M HCl. Deionized water was run through the AAS between samples.

AAS Au Standards.jpg

Plasmids

  • 1 μL DPN1 was added to 45 μL of a solution of K110A (f) and K110A (r) plasmids, made on 2012/10/16. Two of these solutions were created.
  • These two solutions were then placed in a heat block at 37°C for 60 min.


  • 5 μL of each of the two solutions of K110A (f) and K110A (r) plasmids, made on 2012/10/16 were combined with 1 μL of 6X Gel Loading Dye (blue). Then, all 6 μL of each solution was loaded into an agarose gel for gel electrophoresis.
  • Please refer to Keyun Wang's entry for information and observations about preparing and loading the gel, as well as which primers were loaded.