User:Susan Schultz/Notebook/Experimental Biological Chemistry/2011/09/14

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Objective

Re-make and re-measure the absorbance of select BSA standards to create a better regression line.

Prepare growth cultures of ER2566 E.coli with the pMAL-pIII plasmid

Description

The 0.73μL/mL and 1.46μL/mL BSA solutions were prepared with the Bio-Rad protein assay using the same procedure at 9/13/11. They were measured using the same UV-VIS, and the data points replaced the values measured the day before.

The cultures prepared the night before were centrifuged for 15 minutes at 4500rpm at 4°C. Each of the pellets were then re-suspended 1mL of rich broth, and then added to a 1L of rich broth with 100μg/mL ampicillin. The flasks were placed on the shaker/incubator at 37°C at 165rpm. After two and a half hours 0.1mM of IPTG was added to each flask and they were placed back in the same conditions for an additional two and a half hours. The cultures were then removed and centrifuged for 15 minutes at 4500rpm at 4°C. A column buffer was prepared using 20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA. After centrifuging the four pellets were each re-suspended in 50mL of column buffer. They were stored at -20°C

Data

Image:Twentyfour.PNG

Figure 1. Revised regression line of BSA standards

The data point for the 10.22μL/mL was removed to create a better best-fit line.

Notes

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