Team:UCLA/Notebook/Protein Cages/11 August 2015
Phillip's notes:
Introduction: An SDS-PAGE gel will be ran to analyze the results of yesterday’s purification.
Procedures:
All samples were obtained and put on ice.
Using 2x loading dye, 5% BME was added. 142.5uL of 2x dye to 7.5uL of BME.
15uL of each sample was mixed with 15uL of dye. 10uL of dual color ladder was mixed with 10uL of loading dye. Aggregation was observed in the whole cell lysate and in the elution fractions. All samples were boiled for 10 minutes, and centrifuged for 10 seconds to collect at the bottom. The aggregates were noticeably gone.
The SDS-PAGE apparatus was assembled, and 1L of running buffer was made from a 10x solution.
20uL of each sample was added to the wells. The gel was ran at 100V for 30 minutes to ensure even running, then at 120V until the dye front was near the bottom. ~1.5 hours.
The gel was cut out, and stained with coomassie for 1 hour. Destaining took place for 2 hours, with changing the destain buffer every hour.
Conclusions: Since the destain wasn’t completely done, the gel was left in destain on the bench overnight.
Results:
Below is the gel. From left to right: Ladder, ladder, whole cell lysate, unbound proteins, wash 1-3, elution 1-3.
Media:2015-08-12_SDSPAGE_Yeates_PCquad.jpg
It appears that a preliminary purification worked, though more washes can make it cleaner. To avoid the observed aggregation, more lysis buffer may be used next time.
Intro: Transformed Mutant # 10 into chemo-competent BL21-DE3 cells.
Transformation:
Thaw 100 uL cells on ice for 10 minutes. Add 1 uL ligated product to each tube. Incubate on ice for 10 minutes. Rescue cells by transferring them to 4x the cell volume of SOC medium in a 1.5 mL tube. Shake incubate at 37C, 400-600 rpm for one hour. While incubating, pre-warm three agar plates (with proper antibiotic). Spin down cells (4 min at 6,000 rpm). Remove supernatant. Dilute in 110 uL of SOC. Plate 100 uL. Dilute remaining 10 uL in 100 uL SOC. Plate 100 uL. Dilute remaining 10 uL in 90 uL SOC. Plate all of the final dilution. Grow at 37C overnight.
Tyler Lee