User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/17

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Electrophoresis of the mutated DNA plasmids

  • Transferred 5 μL of the mutated plasmid into a new sterilized tube; leaving out one tube containing 45 μL.
  • Simultaneously, added 1 μL of the gel loading dye 6x blue into the 5 μL plasmid and added 1 μL of DpnI into the 45 μL plasmid.
  • The sample containing the DpnI was placed in a VWR analog heatblock at 37°C.
  • The agarose gel was prepared by dissolving 1.23 g of agarose in 35 mL of TAE buffer. The agarose was heated in a microwave for 35s.
  • When the solution is clear, the agarose was poured into the gel electrophoresis tray with the comb in place.
  • A wait period of 30 min. was allotted for the gel to solidify. Upon observing the gel is already in its solidified state, the comb was gently removed to avoid breakage.
  • The chamber containing the gel tray was filled with TAE buffer to the point where the gel is completely submerged of the buffer.
  • The plasmid with the loading dye were inserted to the wells 3 and 4 using an automatic delivery pipet.
  • The Horton 5B Electrophoresis system (chamber containing the tray) was connected to the BioRad power supply powerpac 200 at 85V.

UV-Vis for Chemiluminescence

  • An attempt was made to use the UV-Vis equipment for chemiluminescence by blocking the source lamp (D-2). The measuring mode was set on energy setting. The widest slid width available was 5.0 nm.
  • Data collection was not possible. No signal was collected.

Preparation of Lysozyme stock

  • Michael Nagle prepared a new set of Au/BSA solutions. However, calculations made are not available in his notebook.
  • Lysozyme (14,307 Da) appeared as a white granular solid provided by Sigma. Knowing that 1 Da = 1 g/ mol, the following calculations were made to obtain a stock solution of 15.24 μM.

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