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

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.

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 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.

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.

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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