User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/10/17

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  • to prepare and run Gel Electrophoresis of DNA Primers
  • to prepare and run AA on Au/BSA solutions made yesterday


DNA Gel Electrophoresis

  • The DNA primers from yesterday were removed from the thermo cycler.
  • 5μL were removed from each of the the 50μL in the PCR tubes and placed in separate centrifuge tubes.
  • 1μL Dpn1 was added to each of the remaining 45μL DNA primer.
  • 1μL loading dye was added to each of the 5μL samples.
  • The 46μL samples (containing the Dpn1) were placed in the heat block at 37°C.
  • 1.23 g of Agarose gel was mixed in 35mL TAE buffer and then microwaved for 45s
  • Immediately after it was done microwaving, the gel mixture was poured in to the gel electrophoresis mold and allowed to cool for about 30 minutes, a few minutes after the gel had solidified.
  • After the attachments for the mold (including the well comb) was removed carefully from the gel, TAE buffer was poured over the gel such that the gel was completely immersed in buffer.
  • Then, the DNA ladder was loaded in the first well, with dna primer samples loaded in the other wells. The samples of interest were loaded in wells 3 & 4.
  • The gel electrophoresis was run at 85Volts for an hour.

Centrifuging the Au/BSA mixtures

  • The mixtures were centrifuged in preparation for running AA on the samples.
    • The fibers that are formed during the Au/BSA reactions needed to be formed into a pellet at the bottom of the test tube so that the supernatant was clear of any debris that might clog the AAS.
  • The test tubes containing Au/BSA mixtures were placed in the centrifuge at various rpms, and time periods until the small fibers formed a pellet.
    • 2000 rpm, 5 minutes, 4°C
      • the larger fibers formed a pellet at this setting.
    • Then, 2000 rpm, 10 minutes, 25°C
    • Then, 2500 rpm, 5 minutes, 25°C
    • Then, 3000 rpm, 10 minutes, 25°C
    • Then, 4700 rpm, 15 minutes, 25°C
    • Then, 4700 rpm, 20 minutes, 25°C