Evana Patterson 100750724 Biol2030U Sylvie Bardin 2020-10-10 Lab #2 Purification of Fluorescent Proteins Evana Patterson 100750724 BIOL2030U Sylvie Bardin Evana Patterson 100750724 Biol2030U Sylvie Bardin 2020-10-10 Lab #2- Purification of Florescent Proteins Transformation of Plasmid into E.coli 1.5μl of unknown plasmid DNA (pRSET vectorfp9) contained in a 1.5 mL micro tube was used for the transformation of the unknown plasmid into bacterial strain E.coliJM109-DE3 and placed on ice. After the E.coliJM109-DE3 cells thawed, a pipette was used to extract 50μl of E.coliJM109-DE3 and placed into the microtube containing the plasma DNA (pRSET vector-FP9) and mixed with tip of pipette tip. The remaining E.coliJM109-DE3 was placed back on ice. The resulting mixture was placed on the ice for 30 minutes followed by the heat shock by placing the microtube in a bath of 42◦C for 45 seconds. The microtube was then placed of ice for 2 minutes and 800μl of the LB medium (LB Broth | Biocompare, n.d) was then added to the microtube and the transferred to incubation in a 37◦C hot bath for 45 minutes. The microtube is centrifuged at 8,000rpm for 2 minutes and the supernatant is discarded from the microtube into another microtube. The cells that are re-suspended from the transformation were spread across an LBAmp100 plates (contain the antibiotic ampicillin 100μg/mL). The LBAmp100 plate is then overturned on incubation shelf at 37◦C is kept for 24 hours. Extraction of Protein Using a centrifuge, cytoplasmic proteins (with the fluorescent proteins) resulting in the separation of the cytoplasmic proteins from the cellular debris. 1.4mL of the LBAmp100 kept overnight was placed into a new microtube and then centrifuged. The supernatant is then discarded into beaker. The tube was placed back in the centrifuge at 13,200rpm for 10 seconds, the remaining supernatant was removed. 650 μl of Lysis Buffer (50 mM monobasic sodium phosphate, Lab manual) was added and dissolved in solution by stirring and agitating. Evana Patterson 100750724 Biol2030U Sylvie Bardin 2020-10-10 10% SDS was added and mixed with Lysis Buffer (650 μl). The microtube was then rotated for 30 minutes at room temperature. Lysate supernatant was transferred to new microtube (XI). Purification of Protein Proteins were purified using Nickel-NTA resin affinity chromatography from other cytoplasmic proteins. 130 μl of lysate supernatant (XI) was ultimately transferred to the microtube and kept on ice. -30 μl of the remaining supernatant was stored in freezer at 20◦C. The microtube was then placed on a rotating wheel for 30 minutes and centrifuged at 10,000rpm for two minutes, vortexed. The protein was washed when 60 μl of elution buffer was transferred. The microtube is placed on a rotating wheel for 5 minutes and then centrifuged at 13,200rpm. The new microtube is put into freezer. Identification Using Spectrophotometry 100 μl of Lysis buffer was pipetted into a microplate. . 100 μl of CL sample was placed into the microplate in the well next to the base line buffer. The location of samples is then recorded. A scanning spectrophotometer (Bio-Tek Synergy HT plate reader) read the absorbance of the fluorescent protein in the microtube. Absorption was read over a range of 400nm to 700nm scanned in intervals of 2nm.
