User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/17

Purpose

• To run PCR product on agarose gel for gel electrophoresis
• Digest methylated DNA in PCR plasmid with DNP1
• Determine gold concentration in supernatant of Au/BSA solutions

Procedure

• PCR product for mutation E28K were taken out of thermal controller. 5uL of PCR plasmid were transferred into a separate microcentrifuge tube and is mixed with 1uL of 6X gel loading dye blue from New England bioLabs. 45uL of plasmid were mixed with 1uL of DNP1 and placed on heat block for 1 hour at 37°C.
• 1.23g of agarose mixed with 35mL of 1X TAE buffer and microwaved for 45 seconds
• Agarose with TAE buffer were poured into Gel electrophoresis and solidified after 10 minutes. 1X TAE buffer was poured into gel electrophoresis container.
• DNA ladder is made by mixing 5uL DNA ladder with 1uL of 6X gel loading dye blue. Plasmids with different ADA mutations were loaded in gel in the following order:

                            DNA ladder --> E34K --> E34A --> E34A --> K110A --> K110A --> E34K

• Gel electrophoresis was run at 85 volts for 1 hour.
• Gel was stained in TAE buffer with ethidium bromide. The gel, as well as 20uL of 12.3mg/mL ethidium bromide were added into 100mL of TAE buffer and mixed for 5 minutes. Gel was then transferred to TAE buffer without ethidium bromide to rinse for 5 minutes.
• Gel was observed under UV lamp to see if PCR product has been successfully produced.
• Au/BSA solutions were taken out of incubator. Lids from test tubes and aluminol were taken away from test tubes. The test tubes containing fibers were centrifuged under 7°C for the following conditions:

               2000rpm for 5 minutes, 2000rpm for 10 minutes, 2500rpm for 5 minutes, 3000rpm for 10 minutes, 
               4700rpm for 15 minutes, 4700rpm for 20 minutes

• Some fibers in some solutions still remained on top of solution. The supernatant were taken out using a pipet and placed in 15mL falcon tube.
• Supernatant with different ratios of Au/BSA were run under atomic absorbance spectrophotometer. Blank was done with HCl. Wavelength is excited at 242.8nm. 8 standards were run at the following concentration:

               5ppm, 8ppm, 10ppm, 15ppm, 20ppm, 25ppm, 30ppm, 40ppm

• 12 samples of Au/BSA with various ratios were run in atomic absorbance spectrophotometer after standards were taken.

Notes

• Top of the PCR tubes forms water droplets. The PCR tube containing E34K mutation were centrifuged in minicentrifuge from Thomas Scientific for 1 minute to gather all PCR products together.
• The addition of 1uL of DNP1 enzyme into 45uL plasmid is to digest the methylated DNA from PCR product to make sure all products in solution contain DNA strand produces after PCR.
• Au/BSA solution with some ratios contain heavy fibers floating on top of solution. The solution were centrifuged to separate the fibers from the solutions. Due to unknown speed and time should be used to separate fibers from solutions, different speed and time were performed for successful fiber pellet formation.
• After 4700rpm for 20 minutes of centrifugation, two of the test tubes broke during centrifugation. All samples were taken out, and sample 140Au/BSA and 60Au/BSA were lost as a result. Some samples with fibers remained floating on top were transferred into separate falcon tubes to make sure successfully atomic absorbance spectrophotometer readings.

Results

• Gel was not observed under UV due to the fact that the ladder used was not the right size. Resulting gel shown a smear of DNA ladder about 3cm, and a concentration of dye at the wells of DNA gel, indicating that the gel did not work properly. Picture of gel was not taken.
• The atomic absorbance spectrometer were run on samples, tables is shown below for standard calibration and concentration of gold measured.

Table I: Standard Calibration

Table II: Absorbance for gold in Au/BSA solutions made from 2012/10/16

• Calibration curve was created from standard solutions listed in the first table. Please refer to Melissa's Notebook for graphical representation of the curve. The slope was determine to be 0.0214 abs/ppm. This rate is going to be used to calculate the concentration of gold in Au/BSA fibers. The absorbance of each Au/BSA sample was shown in above table II.