User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/19

Asc Hb Project

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Miniprep of M8S/M33S/M103S/A71M Asc Hb Minipreps were done for 3 colonies of the transformed NovaBlue + M8S/M33S/M103S/A71M. The vacuum protocol was followed: [1] Midiprep of pQE-80-L-Kan A midiprep was done of pQE-80-L-Kan from transformed NovaBlue + QE-80-L-Kan cells. Protocol: [2] Running an Analytical DNA Gel of Ligation Products Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells). When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer. Load 5μL of DNA ladder into the 1st well. Load 5μL of the pQE-80-L-Kan midi-prep from this morning with 1μL 6x loading buffer into the 2nd well (mix before pipetting into well). Load 2.5μL of the wild-type Asc Hb and pQE-80-L-Kan ligation from yesterday with 0.5μL 6x loading buffer into the 3rd well (mix before pipetting into well). Load 2.5μL of the triple mutant (M8S/M33S/M103S) Asc Hb and pQE-80-L-Kan ligation from yesterday with 0.5μL 6x loading buffer into the 4th well (mix before pipetting into well). Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel Place the gel in Ethidium Bromide Stain for about 30 minutes Move the gel to TAE buffer to destain for about 20 minutes View under a UV light Be careful when working with ethidium bromide and UV light. No bands in the lanes with the ligation reactions. I'll see if the transformations grow tonight. Myoglobin Expression Dr. Hartings centrifuged the 50mL starter cultures from yesterday and resuspended the pellets in Terrific Broth with 100μg/mL ampicillin this morning (before 9am). I monitored the absorbance and the OD600 was about 5.6 around 2pm and 6.2 around 2:30pm. I then spun the cells at 4°C at 4500rpm for 15minutes. I resuspended the pellets in 25mL (total) of 25mM Tris, 50mM NaCl, pH 8. I submerged the resuspended pellets in liquid nitrogen, and after they were frozen I stored the cells at -20°C. Transformations of Ligation Products This procedure was done for both ligation products from the overnight ligation (wild-type Asc Hb + pQE-80-L-Kan ligation and triple mutant (M8S/M33S/M103S) Asc Hb + pQE-80-L-Kan ligation): Place plastic culture tubes on ice for about an hour. Place DNA for transformation on ice. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice). Add 1uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute. Add cells (25uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA. Put tube back in ice for 4 minutes. Heat shock at 42°C for 80 seconds. Add 100uL of SOC media. Shake at 37°C for 1 hour. Plate 50uL of culture media on LB/amp plates (prepared fresh today: 1.25g LB (w/o NaCl) + 0.5g NaCl + 1g agar + 50mL dH2O, autoclave liquid cycle, cool to about 60°C, add 50μL 100mg/mL amicillin, pour plate). Incubate inverted overnight at 37°C.