User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/18

Asc Hb Project

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Transformation Results There was no growth on any of the plates from yesterday's transformation. When I removed the microcentrifuge tubes with cells from the incubator shaker, I did notice the cell solutions had begun to turn a bit brownish (which usually is what happens when I leave a tube of leftover cells out overnight). This may be a sign that the cells "went bad" or something in the mixture changed and is affecting them. I thought the tube of cells I used was a new tube of NovaBlue E.coli (not previously defrosted), but maybe not if this happened. I will try the transformations again with another tubes of these cells. This also means we do not have any conclusions on whether or not the PCR from Wednesday was effective or not, since the mini-prepped plasmids should have transformed with no problem. Sonication of Expressed Triple Mutant Asc Hb in E.coli Safety First: Make sure to wear noise blocking earmuffs. Sonicate each tube for 30 seconds at power setting 11. Place each tube on ice for at least 30 seconds after sonication. Repeat this procedure two more times. Distribute sonicated cells between 4 centrifuge tubes for a balanced spin. Centrifuge the cells for 2 hours at 4°C at 18000rpm. Pour off and store supernatent at 4°C. Transformations All of the transformations from yesterday need to be redone. This includes the Asc Hb mutations M33S, M33S/M103S, and M8S/M33S/M103S into NovaBlue E.coli, the wild-type plamids for Asc Hb and myoglobin (wt swMb pT7-7) into NovaBlue E.coli, and the PCR product from two days ago (Asc Hb M8S/M33S/M103S/A71M) into NovaBlue E.coli. The procedure will be the same as yesterday: Take a sterile microcentrifuge tube and place it on ice. Mix 50μL of NovaBlue Competent E.coli with 5μL of plasmid or PCR product in the cold, sterile tube. Incubate this mixture for 30 minutes on ice. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes. Add 200μL of SOC media to the cells/plasmid or PCR product. Shake the mixture at 165rpm at 37°C for one hour. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin. The LB plates were made with 5.25g LB, 4.2g Agar, and 210mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 210μL of 100mg/mL ampicillin was added to it and it was poured approximately evenly into 6 sterile petri dishes. Incubate the plate overnight (inverted) at 37°C.