User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/11/06

Objective

• to run transformations of PCR product into E. coli cells
• to run FPLC on the ADA protein from the third and the fourth of November.

Description

• The cells from the fourth of November were sonciated for 30 seconds and then placed in an ice bath for 30 seconds. This was repeated for a total of 3 times.
• The cells were then transferred to centrifuge tubes, balanced, and placed in the centrifuge at 18,000rpm at 4°C for 2 hours.
• The cells were then filtered using Supor®-450 47mm membrane filter.
• The cells were then run on FPLC, using the binding buffer and elusion buffer from September 26th.
• The cells were collected in 5 mL aliquots and transferred into a Falcon tube.

• The Au/Lysozyme solutions were obtained from the oven. The following images are the results from the Au/Lysozyme reaction.

• The range of Au/Lysozyme ratios, between 20uM ratio to 130 uM ratio.

• A close up of Au/Lysozyme ratios, focusing on the difference between the homologous solutions and the protein fiber solutions.

• UV-Vis was run on the Au/Lyzozyme solutions.

• For the transforamtion of PCR product into E. coli cells, 20μL of PCR DNA and 30μL of E. coli cells were added to a centrifuge tube, in a sterile environment, and then placed in an ice bucket. (This occurred twice, one for each PCR tube.)
• The cells were heat schoked by incubating them in a 42°C water bath for 30 seconds and then placed immediately on ice.
• 250μL of room temperature SOC medium was added to one of the PCR tubes and 250μL of Albumin medium was added to the other PCR tube.
• The cells were shaken at 225 rpm of an hour at 37°C.