User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/11/02

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Objective

To load and run mutated DNA on gel electrophoresis To create and autoclave an LB Agar solution To create AUNP fibers using BSA and HAUCl4.

Procedure

AuNP Sythesis:

New HAuCl4 stock was made. Protocol was followed from the previous week. Three test tubes were used:

Each test tube contained 1 mL of BSA, 1 mL of HAuCl4, and 8 mL of water.

  • Test tube one: Remained in the oven for the full time.
  • Test tube two: Was taken out of the oven every half an hour for ten minutes.
  • Test tube three: Was taken out of the oven every half an hour for ten minutes and then pipetted to mix the solution.

The oven was kept at approximately 70 degrees.

DNA Transformation:

  • Non-methylated DNA was digested with 1 μL Dpnl.
  • LB/agar plates were made using:
    • 0.875 g LB, 0.7 g agar, and 35 mL of water.
  • The mixture was then autoclaved.
  • 35 μL ampicillin was added before the agar completely cooled.
  • The agar was then poured onto the plate and left to solidify.
  • Both a sterile tube and the desired DNA were placed in an ice bucket for 15 minutes.
  • 5 μL of DNA and 30 μL of cells were added to the bottom of the sterile tube, then incubated on ice for 30 minutes.
  • The DNA/cells were then heat shocked at 42°C for 30 s, and incubated on ice for 5 minutes.
  • 250 μL of SOC media was added to the tube, which was then incubated in the shaker at 37°C for 1 hr.
  • 100 μL of the cells were spread on the LB/agar plate using sterile technique.
  • The plate was then stored inverted overnight in an oven at 37°C oven.

Electrophoresis:

  • a 1% agarose gel was made by adding 0.25 g of a 0.01 g/mL agarose solution to 25 mL of a 1x tris base, acetic acid, and TAE buffer. This mixture was then microwaved for 40 seconds.
  • 2 μL of 6x gel loading dye was mixed with 10 μL of the DNA sample.
  • A ladder was prepared containing DNA, glycerol, and gel loading dye.
  • Each DNA solution was loaded and the gel was run at 80 V for 40 minutes.
  • The gel was removed and placed in TAE and EtBr solution, then mixed for 15 minutes.
  • The gel was rinsed for 5 minutes with TAE.

Results

Each test tube formed purple fibers. Test tube one's fibers were long, while the other two test tubes had shorts more aggregated fiber clumps.

Notes

AuNP:

  • T=0 occured at 12:30 pm.
  • The concentration of BSA was 1.55 microM. The concentration of HAuCl4 was 2.5 mM.


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