MIT OpenCourseWare http://ocw.mit.edu5.36 Biochemistry LaboratorySpring 2009For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.List of Abbreviations: Abl (or c-Abl) Abelson kinase Abl(229-511) the Abelson kinase domain, which is constitutively active for kinase activity when expressed as an isolated domain APS ammonium persulfate bp base pairs EtBr ethidium bromide IPTG Isopropyl-ß-D-thiogalactopyranosid kan kanamycin, an antibiotic kb kilobase pairs MW molecular weight (reported in g/mol) OD600 optical density (absorbance) at 600 nm RE restriction enzyme strep streptomycin, an antibiotic TAE tris acetate electrophoresis TBS tris buffered saline Tm melting temperature TEMED etramethylethylenediamine wt wild type w/v weight/volume 48APPENDIX A: Common biochemistry laboratory procedures used multiple times in URIECA modules 4 and 5. A1: Bio-Rad assay for quantifying protein concentrations http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth/biorad_assay.pdf First prepare bovine serum albumin (BSA) solutions to use as standards. To prepare 5 mL of a 1 mg/mL aqueous solution of BSA, weigh out 5 mg of BSA into a 15-mL conical tube, add 5 mL of water, and invert or vortex the tube to completely dissolve the BSA. BSA aliquots may be stored long term at -20 °C. Prior to each assay, prepare five dilutions of the BSA stock solution to create a standard curve. The linear range of the Bio-Rad protein assay is 0.2 to 0.9 mg/mL. Therefore in six separate 0.65-mL eppendorf tubes, label and add the following: 270 μL of the BSA stock and 30 μL of deionized water (to give 0.9 mg/mL), 210 μL of BSA and 90 μL of water (to give 0.7 mg/mL), 165 μL of BSA and 135 μL of water (to give 0.55 mg/mL), 120 μL of BSA and 180 μL of water (to give 0.4 mg.mL), 60 μL of BSA and 240 μL of water (to give 0.2 mg/mL), and no BSA and 300 uL of water (to use as your “blank”). Prepare 20 mL of the assay dye reagent by diluting one part Bio-Rad dye concentrate with four parts DI water. Filter the resulting solution and store any unused reagent for up to two weeks at room temperature. To create a standard curve using the BSA standards, pipette 20 μL of each standard into an eppendorf tube. Add 980 µL of the prepared dye reagent to each tube and vortex briefly. Incubate the tubes at room temperature for 5 to 50 minutes. Measure the absorbance of each solution at 595 nm by UV/Vis spectrometry using disposable polystyrene cuvettes. The sample with 20 μL of water in 980 µL of assay reagent should be used as your “blank” in the spectrophotometer. Plot the absorbance vs. concentration for the BSA standards using Microsoft Excel or another graphing program and determine the R-squared value and slope of the best-fit line to establish that you can obtain a strait line with your standards. If your R-squared value is less than 0.9, repeat the exercise until you achieve an acceptable line. Good laboratory practice calls for the creation of a new standard curve each time you run a protein assay. To determine the concentration of unknown samples, combine 20 uL of your sample (or an appropriate dilution) with 980 µL of assay reagent and measure the absorbance at 595. Use the slope of the standard curve to calculate the sample concentration in mg/mL. A2: Varian Cary 100 Spectrophotometer Instructions 1. Turn on the spectrophotometer. 2. Open the “simple reads” program, found as a desktop icon or under the Cary WinUV menu under the “Start” button. 3. The lamps are on if under the “Commands” menu “Lamps Off” is displayed. If “Lamps On” is displayed, click on it to turn on the lamps. For accurate measurements, allow the lamp to warm up for at least 15 minutes before use. 4. To set the instrument, click the “Setup…” command button on the left side of the screen. In the “Read the wavelength” scroll box, type in the desired wavelength. 49The “Abs” button in “Y mode” should be selected. When the setup is complete, click the “OK” button to exit the window. 5. Prior to measuring the absorbance of your samples, you must zero the instrument. To do this, open the lid and determine which cell is in the light path. Insert a cuvette with your blanking solution in the instrument. Close the lid completely, and press the “Zero” command button on the left side of the screen to zero the instrument at the chosen wavelength. The blank should now have a absorbance read of zero give or take the small amount of “noise” present at all times. 6. You are now ready to measure the absorbance of your samples. Insert a cuvette containing a sample into the instrument, close the lid completely, and press the “Read” button at the top of the screen. Record the absorbance measurement. 7. Under the “File” menu, select “Print” to print your data. 8. When finished, close the program and return to the desktop. 9. Turn off the lamps, then turn off the instrument. A3: DNA isolation from a bacterial pellet (miniprep) http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1043788_HB_QIAprep_122006.pdf Miniprep Procedure: a.) Harvest the bacterial cells from your 3-mL overnight culture by transferring 1.5 mL into a 1.5 mL eppendorf tube and spinning down the cells in a microcentrifuge for 3 minutes. Discard the supernatant and add the remaining 1.5 mL of cell culture to the tube. Repeat the centrifugation and discard the supernatant. You should have a small bacterial pellet at the bottom of the tube. b.) Check that RNaseA has been added to Buffer P1. Add 250 μL of Buffer P1 to the cell pellet and completely resuspend the pellet by vortexing. c.) Add 250 μL of Buffer P2 and mix by inverting the tube 4-6 times. Do not vortex, since that can cause shearing of the DNA. If you have properly lysed the cells, the cell suspension will turn blue after the addition of the P2 buffer. If there are colorless regions or brown clumps in the cells, continue mixing until a homogenous blue solution appears. d.) Add 350 μL of Buffer N3 and mix immediately by inverting the tube 4-6 times. The solution should become colorless and cloudy. e.) Centrifuge the tube for 10 min at 13,000 rpm in you bench top microcentrifuge. A compact white pellet should form. f.) Apply the resulting supernatant (which contains the plasmid DNA) to a QIAprep spin