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.SESSION 3 (lab open 1-5 pm) During sessions 3 and 4 you will verify that the plasmid DNA you isolated contains a construct of the expected size for the Abl kinase domain. You will then design primers for subsequent site-directed mutagenesis. In preparation for purifying the H396P Abl kinase domain, you will prepare all the necessary buffers for the lysis and purification. In session 4, you will also prepare a standard curve for future protein quantification. Restriction enzymes (RE’s) are enzymes that selectively cleave double stranded DNA based on recognition of specific, short DNA sequences called recognition sites. In DNA engineering, restriction enzymes are used to cut open plasmids for the ligation of a complementarily-cut DNA insert into the plasmid for protein expression. In Session 2, you isolated a DNA plasmid (the pET-28a vector) containing an insert encoding residues 229-511 of the Abl kinase domain. Since the gene encoding the Abl kinase domain is positioned in the pET-28a vector between Xho1 and an Nde1 recognition sites, treatment of the plasmid with those two restriction enzymes should result in excision of the ABL insert. Today you will excise the insert by digestion of the isolated plamid DNA with Xho1 and Nde and check the size of the digestion products on an agarose gel to confirm that your vector and insert are the expected size in base pairs (bp). As denoted in the vector map, the pET28a vector (minus the segment between the Nde1 and Xho1 sites) is 5,289 bp, and the ABL insert should be 849 bp. As experimental controls, you will simultaneously prepare a no-enzyme “mock” digest and digests containing only one of the two restriction enzymes required to excise the ABL insert. By gel analysis of the four reactions, you will expect to see a band for the no-enzyme “mock” reaction at approximately 6,138 bp (consistent with the size of the insert plus vector. The one-enzyme reactions should linearize the plasmid DNA without excising any segment. Since coiled plasmid DNA travels further by electrophoresis than equivalent linearized DNA, these reactions should result in bands with an apparent size slightly larger than the uncut plasmid. A sample gel is provided below. 12345lane 1 500-bp DNA ladderlane 2 no-enzyme "digestion"lane 3 Xho1-only digestionlane 4 Nde1-only digestionlane 5 Xho1/Nde1 digestion500 bp1000 bp1500 bp2000 bp 131.) DNA digestion with Xho1 and Nde1 restriction enzymes Set up analytical-scale digests of your plasmid DNA using 1 μg of DNA and final reaction volumes of 20 μL each. In each of four sterile 0.65 mL tubes, add the following components in the order listed, where x = the volume of miniprep elution added (in μL) to give 1 μg of DNA: no enzyme Xho1 only Nde1 only Xho1/Nde1 Sterile water 17.8 – x μL 17.3 – x μL 17.3 – x μL 16.8 – x μL RE 10X Buffer D 2 μL 2 μL 2 μL 2 μL BSA, 10 μg/ μL 0.2 μL 0.2 μL 0.2 μL 0.2 μL Plasmid DNA, 1 μg x μL x μL x μL x μL Mix by pipetting, then add: Xho1, 10 units/ μL 0 μL 0.5 μL 0 μL 0.5 μL Nde1, 10 units/ μL 0 μL 0 μL 0.5 μL 0.5 μL ______ ______ ______ ______ Final volume 20 μL 20 μL 20 μL 20 μL Mix the reactions gently by pipetting, close the tubes, and centrifuge for several seconds in a bench-top microcentrifuge. Incubate the digestions for 1 hour in the 37 ºC incubator. Meanwhile, prepare an agarose gel (described below) to analyze the digested DNA by electrophoresis. When the digestions are complete, remove the tubes from the incubator and add 4 μL of 6X nucleic acid loading buffer to each of the reactions. DNA electrophoresis enables size-based DNA separation. Due to the negatively charged phosphate backbone of DNA, DNA molecules migrate from the negatively-charged to the positively-charged end of the gel. The smaller molecule travel faster than the larger molecules. The size (in bp) of each separated DNA fragment is determined by comparison to a DNA ladder that has a band at given intervals of bp. Here we will use a 500 bp ladder that includes bands of DNA that are 500 bp, 1000 bp, 1500 bp, 2000 bp, and so on. Depending on the size of the DNA fragments you wish to analyze, more of less agarose can be incorporated into a DNA gel. The higher the percentage of agarose, the smaller the effective range of separation will be. For analyzing the 849-bp insert and the 6.1 kb Abl-containing vector, you will prepare a 1.2% (w/v) agarose gel. 2.) Preparation of a DNA 1.2 % agarose gel Set up the gel box as demonstrated by the TA. Insert the gel comb at the negatively-charged (black) end of the box. In a 200-mL Erlenmeyer flask, weigh out 1.2 g agarose and add 100 mL of 1X Tris acetate electrophoresis (TAE) buffer. To prepare the 1X buffer, dilute 10 mL of 10X TAE (provided by your TA) with 90 mL of DI water. Dissolve the agarose by heating the flask in a microwave on medium heat until the solution just boils. Prior to microwaving, plug the top of the flask with a paper towel to help prevent boiling over. All of the agarose solid should be dissolved and the solution should be clear. Cool the solution to approximately 60 ºC (cool enough to hold the flask 14comfortably), and pour the solution into a prepared gel box. Allow the gel to set for approximately 30 minutes before loading. The colorless gel solution will have a bluish tint when the gel has solidified. Once the gel has solidified, carefully remove the comb by pulling strait upwards. Pour 1X TAE into the gel box so that it just covers the gel. Using a gel-loading tip that fits the 20-μL pipette, load 3 μL of 500-bp DNA ladder and 10 μL of each of your four DNA digestion samples. Remember, DNA will migrate toward the positive (red) electrode. Loading the samples at the wrong end will cause the samples to run off the gel! Place the top on the DNA gel box and run the gel at 100 Volts for 1 hour. Once the gel running is complete, carefully remove the gel from the box and place it in a small plastic container, such as the top of a gel-tip box. Stain the gel with approximately 0.5 μg/ mL ethidium bromide (EtBr) solution for 10 minutes by agitating on a gel rocker. EtBr is a fluorescent DNA intercalating agent, which means it inserts itself