Plasmid Challenge: Restriction Digest and Electrophoresis

    Restriction DigestPlasmids are typically small independently replicating extrachromosmal DNA that can be found in bacteria and some protozoans. Most plasmids are several kilobases in size; therefore researchers can confirm the identity of a plasmid and/or the presence of an insert (gene) using a technique known as restriction digest.  Restriction digest takes advantage of special enzymes that can cut (or digest) DNA at specific sequences. If the sequence of the plasmid is known, then restriction enzyme sites can be identified and sizes of DNA fragments can be predicted. Folliwng digestion, electrophoresis is used to visualize the fragments and identify the plasmids. Plasmids are sold commercially and often used in a process known as genetic cloning.  Transformation introduces the plasmid into living bacterial cells so that the gene of interest can be replicated. Heat shock transformation and electroporation are the two methods of bacterial transformation commonly used in the laboratory. Both methods require competent cells, which are bacterial cells that can take up DNA. Competent cells may be prepared in the laboratory or purchased commercially. In effort to identify successful transformation, plasmids are often engineered to contain antibiotic resistant genes. This helps ensure that all bacteria present contain the gene of interest and that it has been successfully replicated. In this lab, you will be given two different plasmids of unknown identity. Your task is to identify the each plasmid based on restriction digest and electrophoresis, then test your hypothesis by transforming the plasmid of your choice into E. coli. If transformation is successful, you will be able to isolate and measure the protein gene product. Part 1: Pouring Agarose gels 1. Prepare 75 ml of 1% agarose solution in 1X TAE in an Erlenmeyer flask.  To calculate the amount of agarose you need, use the formula: 1% agarose solution = 1 gram agarose in 100 ml buffer. 75 ml of 1% agarose solution = _______ grams of agarose  + ______ ml of 1X TAE 2. Place your agarose and buffer into your Erlenmeyer flask and swirl to mix.3. Place your solution on a hot plate/microwave at your lab bench. 4. Heat on high, occasionally swirling (be sure to wear the heat guard glove).  Heat the mixture until boiling and transparent.  Be careful not to let your solution boil over! 5. Carefully remove your boiling solution from the hot plate/microwave using hot guard gloves and place your agarose solution on the bench top. 6. Once your solution is cooled to ~60°C (to touch), you may carefully and slowly pour your gel on into the gel case. 7. Do not disturb the gel until it has solidified. Part 2: Restriction Digest1. Label 4 microfuge tubes 1-4.  2. Setup the following reactions in each tube a. add water first and enzyme last directly to reaction mixtureb. switch tips every time!Tube 10X Buffer Water DNA BamHI EcoRI1 2 µl 12 µl 5 µl Plasmid A 1 µl -----2 2 µl 12 µl 5 µl Plasmid A ----- 1 µl3 2 µl 12 µl 5 µl Plasmid B 1 µl ------4 2 µl 12 µl 5 µl Plasmid B ----- 1 µl 3. Briefly spin down the tubes in a centrifuge for ~30 seconds.4. Incubate at 37°C in a heat block or water thermostat for 5 min   Part 3: Gel Electrophoresis1. Make certain your gel has solidified before proceeding with this section.2. Add 5 µl of DNA loading dye to all samples.a. SWITCH TIPS3. Vortex and spin if necessary4. Load 20 µl of your sample into each well of the gel using the table below  5. Run the gel at 100V for ~20 minutes.     Lane Sample1 Ladder2 Plasmid A + BAMHI3 Plasmid A +EcoRI4 Plasmid B + BAMHI5 Plasmid B +EcoRI   Size: 5371bp Part 4: Predict the number and size for the fragments (in bp) expected for each restriction digest reaction: Plasmid EcoRI BamHIpGLO PRFP Post Lab due next lab meeting (it attached to the order) Summarize the results of your restriction digest and predict the identity of plasmid A&B. This should be no less than 1 paragraph                                 pGLO Plasmid Transformation and Analysis   1.  Label 2 sterile microfuge tubes: + p - p 2.  Add 250µl of ice cold CaCl2 (calcium chloride) solution to each tube.3.  Obtain a streak plate of E. coli from the instructor.4.  Using a sterile loop, pick 4 - 5 isolated colonies from the plate, then add them directly to the cold CaCl2 solution in the +p tube.  Twist the toothpick vigorously to dislodge the cells from the loop. Repeat for the –p tube.  NOTE: Try to avoid scraping up agar when you are picking colonies.5.  Be sure that the cells are completely re-suspended in each tube by tapping and mixing. Note: You should not have chunks of colonies floating around6.  To the tube labeled “+ p” add 5 µl of your selected plasmid DNA from Part 1.  I have added 5 µl of your selected plasmid DNA from Part 1 To the tube labeled “- p” as well by mistake   (It is good for the discussion) Note: Be sure to add the plasmid directly to the cell suspension.7.  Incubate both tubes on ice for 10 minutes.While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom: • Label one LB/amp plate: + p• Label the LB/amp/ara plate: + p• Label the other LB/amp plate: - p• Label the LB plate: - p 8.  Obtain your tubes from the ice and place both tubes at 42°C for exactly 50 seconds9.  Return both tubes immediately to the ice bucket for two minutes.10.  Add 250µl Luria Broth (“LB broth”) to each tube and mix.  Be sure to change tips in between tubes.11.  Incubate the cells for 10 minutes at room temperature.12.  After your incubation is complete, you are ready to spread your cells onto the plate.  13.  Add 100µl of your “– p” transformation mixture to each of the appropriate plates.  Spread each plate one at a time with a sterile spreader.  Your instructor will demonstrate this technique to you.  14.  Repeat the process for your “+ p” transformation mixture.15.  Once you have spread your transformation mixtures on all 4 plates, let the agar plates sit on the bench for ~5 minutes.16.  Stack the agar plates lid side down and secure with paper tape containing your group’s initials.  Place the plates in the 37°C dry incubator overnight. 17. You will have to come and check on your bacteria within 48 hours using the fluorescent light Post Lab (it attached to the order) Analyze your results in 1-2 paragraphs.  Be sure to consult references to explain your data. Your response should include: the impact of your plasmid on your transformation; whether you selected pGLO or pRFP based on the electrophoresis results; why some plates glowed and others did not based on the growth environment; if the color of your fluorescene matched your expectation-why or why not.