Lambda (λ) DNA Restriction Digest and Electrophoresis Lab

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Donald Taylor
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Lambda (λ) DNA Restriction Digest and Electrophoresis Lab Procedure DAY ONE: restriction digestion Today we will be exposing the lambda DNA to restriction enzymes.

1 Lambda (λ) DNA Restriction Digest and Electrophoresis Lab Procedure DAY ONE: restriction digestion Today we will be exposing the lambda DNA to restriction enzymes. For background knowledge, make sure you can answer the following questions: 1. What are restriction enzymes? 2. Why are restriction enzymes are necessary before DNA can be electrophoresed? PROCEDURE Read very carefully!! Safety Notes: Please use pipets properly; do not point or release the tip at another person Wear safety goggles to avoid exposure of enzymes to your eyes Perform the day 1 procedure by following the direction below. AS YOU PROCEED THROUGH THE PROCEDURE, create a table like the one below in your lab book in which you summarize the procedural step (note, you must summarize, please do not write every word) and briefly explain WHY this step must be performed in order to have a successful restriction digest and electrophoresis of DNA. If you don t know why you are doing something, ask each other and then your teacher for clarification. Procedure 1. Obtain micro test tubes that contain lambda DNA (λ), each enzyme (PstI, HindIII and EcoRI) and restriction buffer (RB). Keep all these tubes on ice. Explanation 2. Centrifuge the tubes for about 3 seconds. Be sure your centrifuge is balanced. 3. Obtain and label 4 micro-test tubes: a. L: for lambda DNA without any added enzymes b. P: for lambda DNA and the restriction enzyme PstI c. E: for lambda DNA and the restriction enzyme EcoRI d. H: for lambda DNA and the restriction enzyme HindIII

2 4. Add reagents in the order given in the table below. Use a new pipette tip each time you use the pipette! Note: these volumes are TINY! You may feel like you are hardly pipetting anything! Refer to your pipet practice mini lab for a sense of just how small these volumes are. 5. Mix tube contents gently by centrifuging for about 3 seconds. Be sure your centrifuge is balanced. 6. Label a foam micro-tube holder with your group initials. Put in your tubes and return them to your teacher. The tubes will sit at room temperature overnight. 7. Once you are done with the procedure, please clean your lab station and properly put away your safety goggles. Used pipet tips and the original micro tubes of lambda and each enzyme should go in the trash. Obtain a stamp from your teacher when you are done. Stamp Table 1: Solutions added to each micro tube in procedure step 3 Add to Tube: Tube: L P E H Restriction Buffer 6 l 5 l 5 l 5 l Uncut DNA 4 l 4 l 4 l 4 l Enzyme PstI -- 1 l Enzyme EcoR I l -- Enzyme Hind III l Total Volume 10 l 10 l 10 l 10 l Procedure DAY TWO: gel electrophoresis Today we will be running the lambda DNA through an electrophoresis gel. For background knowledge, make sure you can answer the following questions: 1. Why does DNA move through an electrophoresis gel? 2. Compare the rate of travel of DNA fragments of different sizes. Safety Notes: Please use pipets properly; do not point or release the tip at another person Wear safety goggles to avoid exposure of enzymes to your eyes Avoid getting the loading dye and DNA stain on your hands and clothes.

3 You must work efficiently and quickly during the first part of the lab. You should have your gels running within 15 minutes of the start of class. That will enable the DNA to be in the electrophoresis chamber for as long as possible. Perform the day 2 procedure by following the direction below. PERFORM STEPS 7-15 FIRST, BEFORE YOU WRITE AND EXPLAIN THE PROCEDURE. Then, when the gel is running (step 15), create a table like the one below in your lab book in which you summarize the procedural step (note, you must summarize, please do not write every word) and briefly explain WHY this step must be performed in order to have a successful restriction digest and electrophoresis of DNA. If you don t know why you are doing something, ask each other and then your teacher for clarification. Procedure 1. Obtain your micro-tubes from yesterday and the DNA marker (M) from your teacher. The marker is lambda DNA that has already been cut with HindII to which we can compare our results. Explanation 2. Add 2 l of sample loading dye (LD) into each tube (L, P, E, H and M) 3. Mix tube contents gently by centrifuging for about 3 seconds. Be sure your centrifuge is balanced. 4. Heat your samples and the marker at 65 C for 5 minutes and then put them on ice. While you are waiting, set up your gel boxes as described in steps 11 and Place the agarose gel into the electrophoresis tray. Be sure you have the wells of the gel oriented towards the negative (black electrode) end of the tray. 6. Fill the electrophoresis chamber and cover the gel with 0.25X TAE buffer. This will be about 275 ml of buffer, but the exact amount will vary. You just want to add enough to completely cover the top of the gel with a layer of buffer.

4 7. Using a separate tip for each sample, load 10 l of each sample (M, L, P, E, H) into separate wells in the gel chamber. Be sure you make note of the order in which you added the samples. 8. Carefully place the lid on the electrophoresis chamber. Connect the electrical leads into the power supply (red to read and black to black). 9. Your teacher will help you turn on the power box so you can run the gel at 200V. 10. When there are 10 minutes left in class, turn off the power and remove the top of the gel chamber. Carefully remove the gel tray from the box. Be careful the gel is very slippery. Slide the gel into a staining tray that has been labeled with your groups initials. You will share a tray with another group, so each group should label one end of the tray. 11. Add enough 1X DNA stain to cover the gel. Let the gels stain overnight. 12. Once you are done with the procedure, please clean your lab station and properly put away your safety goggles. Used pipet tips should go in the trash. Obtain a stamp from your teacher when you are done. Stamp

5 Procedure DAY THREE: viewing results Safety Notes: Avoid getting the DNA stain on your hands and clothes. 1. Pour off the DNA stain into the sink 2. Carefully view your gel using a light source from beneath 3. Record your results by copying the locations of the bands of DNA and the wells onto a clear sheet of acetate with a permanent marker. If you have a group member absent today, please make an acetate copy for them as well. 4. Once you are done with the procedure, please clean your lab station. Stamp QUALITATIVE ANALYSIS 1. Attach the plastic sheet tracing of the banding patterns from the DNA gel electrophoresis into your lab book. Be sure it is labeled with a title and a listing of which sample was in which well. 2. Attach a copy of the idealized results into your lab book. Be sure it is labeled with a title and a listing of which sample was in which well. 3. Create a table comparing the number of bands depicted in your hypothesized results, the actual results and the idealized results for EACH enzyme sample. 4. Why do you think there is a difference between the hypothesized, actual and ideal results? QUANTITATIVE ANALYSIS USE IDEALIZED RESULTS 5. Create a data table like the example below in your lab book. Copy the actual size (bp) of the marker sample given in the table. For now, leave the other rows blank.

6 6. Using a ruler, measure the migration distance of each band for each DNA sample. Measure the distance in millimeters from the bottom of the loading well to each center of each DNA band and record your numbers in the table. If your marker sample does not have six bands, then fill in the distanced for as many bands that your gel has, from largest band to smallest. Not all samples will have six bands. 7. The number of base pairs in each of the DNA fragments on your gel can be determined by graphing the size of the known fragments (the marker sample fragments) against the distance each DNA band moved through the gel. This is called a standard curve. This is most conveniently done on semi log graph paper. Using the semi-log graph paper provided, plot distance traveled (X-axis) versus size for bands 2 6 of the size marker sample. Tape your graph into your lab book. Be sure it has a title and labeled axis. 8. Use a ruler and draw a best fit line for the points. Extend the line all the way to the right hand edge of the graph. 9. To estimate the size of the other fragments, find the distance that fragment traveled. Locate that distance on the x-axis of your standard graph. From that position on the x-axis, read up to the standard line, and then follow the graph line to over to the y-axis. You might want to draw a light pencil mark from the x-axis up to the standard curve and over to the y-axis showing what you ve done. Where the graph line meets the y-axis, this is the approximate size of your DNA fragment. Do this for the L, P, E and H samples. Record the approximate size (in bp) of each band of DNA in the data table. REFLECTION Answer the following questions in complete sentences in your lab book: 1. How does the application of DNA electrophoresis relate to your understanding of DNA structure? 2. What did you learn by doing this lab?

Objectives Introduction restriction endonucleases Examples: Hind III: Eco RI: Pst I:

Objectives Introduction restriction endonucleases Examples: Hind III: Eco RI: Pst I: Objectives Before doing this lab you should understand how gel electrophoresis separates DNA molecules present in a mixture and how restriction endonucleases function. After doing this lab you should be