User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/05

Asc Hb Project

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Bradford Protein Assay Standards were set-up in the following way in plastic cuvettes: Standard Amount of Bradford Dye Amount of 14.6μg/mL BSA Amount of Distilled Water Final Concentration in Cuvette Blank 200 μL 0 μL 800 μL 0 μg/mL Standard 1 200 μL 50 μL 750 μL 0.73 μg/mL Standard 2 200 μL 100 μL 700 μL 1.46 μg/mL Standard 3 200μL 250μL 550μL 3.65μg/mL Standard 4 200μL 400μL 400μL 5.84μg/mL Standard 5 200μL 550μL 250μL 8.03μg/mL Standard 6 200μL 700μL 100μL 10.22μg/mL The BSA was diluted from 1.46mg/mL to 14.6μg/mL by adding 5μL of 1.46mg/mL BSA to 495μL of distilled water. Then, when more BSA was needed, 10μL of 1.46mg/mL BSA was added to 990μL of distilled water. And when more was needed one more time, 10μL of 1.46mg/mL BSA was added to 990μL of distilled water. Spectra from 200nm-800nm was taken of each of the standards and Blank. The wavelength recorded at 595nm was used to create a standard curve for protein concentration. The triple mutant Asc Hb protein at its unknown concentration was diluted by a factor of 100 by combining 5μL of the protein with 495μL distilled water. (this was the protein concentrated on 5/22/12). This was done for each of the tubes the concentrated protein was in (Tubes 1,2,3). 100μL of each 100x dilute protein was then mixed with 700μL of distilled water and 200μL of Bradford dye in a plastic cuvette. Spectra of these mixtures was taken from 200nm-800nm. 50μL of each 100x dilute protein was then mixed with 750μL of distilled water and 200μL of Bradford dye in a plastic cuvette. Spectra of these mixtures was taken from 200nm-800nm. 20μL of the Tube1 100x dilute protein was then mixed with 780μL of distilled water and 200μL of Bradford dye in a plastic cuvette. A spectrum of this mixture was taken from 200nm-800nm. Note: This tube looked like it had a more concentrated protein in it than the others (darker color). Bradford Protein Assay Results The absorbance from the blank (800μL distilled water with 200μL Bradford dye) was subtracted from the absorbances of the standards at 595nm to create the standard curve to determine protein concentration. The absorbance of the blank was 0.571. Standard Concentration Absorbance Corrected Absorbance Standard 1 0.73 μg/mL 0.627 0.056 Standard 2 1.46 μg/mL 0.684 0.113 Standard 3 3.65μg/mL 0.876 0.305 Standard 4 5.84μg/mL 0.919 0.348 Standard 5 8.03μg/mL 1.053 0.482 Standard 6 10.22μg/mL 1.167 0.596 The equation of this line was determined by Excel to be (forced through the origin) y=0.0608x. The absorbance of the blank was also subtracted from the Asc Hb triple mutant protein absorbances at 595nm. The concentration of the protein undilute was determined by dividing the corrected absorbance by 0.0608 and then multiplying that quotient by the dilution factor. The absorbances, corrected absorbances, and calculated concentrations of the triple mutant Asc Hb are as follows: Tube Number Dilution Factor Absorbance Corrected Absorbance Calculated Concentration (μg/mL) Calculated Concentration (mg/mL) Tube 1 1000 1.744 1.173 19292.76 19.29276316 Tube 1 2000 1.29 0.719 23651.31579 23.65131579 Tube 1 5000 0.865 0.294 24177.63158 24.17763158 Tube 2 1000 1.104 0.533 8766.447368 8.766447368 Tube 2 2000 0.883 0.312 10263.15789 10.26315789 Tube 3 1000 1.057 0.468 7993.421053 7.993421053 Tube 3 2000 0.841 0.27 8881.578947 8.881578947 Preparation for Making Competent Cells A new protocol will be used to make M15 cells competent. The procedure can be found on this webpage: [[1]]. To prepare for this procedure a few things were made: 100mL LB: 2.5g LB + 100mL distilled water, autoclave on liquid cycle 100mL TFB1: 1.21g RbCl + 0.99g MnCl2 + 0.29g Potassium Acetate + 15mL glycerol, pH to 5.8 with HCl and KOH, dilute to 100mL with dH2O (final concentrations: 100 mM RbCl, 50 mM MnCl2, 30 mM Potassium acetate, 10 mM CaCl2, 15% glycerol) This solution will need to be sterile-filtered before use 50mL TFB2: 0.12g HEPES + 0.06g RbCl + 0.41g CaCl2 + 15mL glycerol, pH to 6.8 with KOH, dilute to 50mL with dH2O (final concentrations: 10 mM HEPES, 10 mM RbCl, 75 mM CaCl2, 15% glycerol) This solution was sterile-filtered today Start of Growth for Making Cells Competent Take one colony from a plate that was previously streaked out with M15MA E.coli with a sterile wooden stick. Swirl the stick around in 10mL of LB in a sterile test tube. Place the test tube at 37°C at 150 rpm to grow overnight (~16 hours).