E. Coli Library Bacteria Report

Materials and Methods

Cloning E.coli Library Bacteria and Mysterious Bacteria

The gradients of the media used for cloning E. coli bacteria included nutrient agar, ampicillin and arabinose sugar; X-gal was an extra gradient in unknown bacteria media. X-gal is an organic compound that consists of galactose linked to a substituted indole, it is used to detect β – galactosidase activity by yielding a blue compound. A purified E. coli library bacteria, containing a DNA fragment of the pG10 plasmid, was added directly on an agar plate and streaked using a sterile inoculation loop. According to the cloning protocol of unknown bacteria stock, 10 µL unknown bacteria was added to 90 µL nutrients media, and 10 – fold serial dilution were used to accurately

Prior to loading any protein sample, HIC was washed using four different solutions including equilibrium buffer (a highly salt solution 2M (NH4)2SO4), binding buffer (a very high salt solution 4M (NH4)2SO4), Wash buffer (a medium salt solution 1.3M (NH4)2SO4), and TE (elution) buffer (a very low salt solution 10 mM Tris/EDTA). According to the protein chromatography protocol, to prepare the chromatography column, the HIC column shook vigorously to resuspend the beads, the top cap was removed, and the bottom tap was snapped off, allowing the liquid buffer to drain (took 3-5 minutes). Then 2 mL of equilibrium buffer (i.e. 1 mL at a time) was added. The equilibrium buffer was allowed to drain until it reached the 1 mL mark above the white beads, then the column was recapped at each side and stored at room temperature for further

In a 0.5 ml vial, a 4µl purified protein combined with 2µl denaturing solution, and ladder was prepared by adding 6µl aliquot of protein 80. Both purified protein samples and the ladder were heated at 95oC for 5 minutes, then wait until it cool down and spun it for 15 seconds. Then, an 84µl deionized water to samples and the ladder and vortex.

After preparing the samples and the ladder, the base-plate of the chip priming station was adjusted to position A and the syringe clip to its middle position. A new chip protein was unsealed from its bag and placed on the chip priming station. A 12µl of gel-dye mix was pipetted in the well-marked G. A plunger at 1 ml was placed and the chip priming station was closed. The plunger was pressed until held by clip followed by waiting period of 60 seconds, then the clip was releases followed by 5 seconds waiting period. Slowly, the plunger was pulled back into 1 ml. Then all the wells labeled with "G" was loaded with 12µl of gel-dye mix, and the well labeled DS was loaded with 12µl of destaining