RESTRICTION OF LAMBDA DNA
Lambda DNA comes from a virus called Phage Lambda. This virus is harmless to man and therefore makes an excellent source of DNA. Below is a map of some of the genes found on Lambda.
Lambda is approximately 48,000 base pairs long. Since Lambda is a virus, it is able to express some of its genes by taking over the bacterial cell that it infects. In this investigation, you will observe the effects of three restriction enzymes on Lambda DNA. A restriction enzyme (also known as an endonuclease) will search for a specific sequence of base pairs. It will cut (chemically separate) a piece of DNA at that specific arrangement of base pairs. The DNA arrangement may appear many times, thereby providing the fragments that we are able to separate. When different restriction enzymes are used to cut a single strand of DNA such as the Lambda DNA, fragments of varying sizes are produced. These fragments can be separated by their size/shape through gel electrophoresis. DNA is negatively charged due to its phophate backbone. Therefore, DNA will move toward the positive electrode. Gel electrophoresis is based on the principle that the rate a molecule moves through a gel is determined by its size and/or shape. Thus, a small fragment will be able to move quickly, whereas a large fragment will move more slowly. By the same token, a DNA piece that is extended and very long may have more difficulty moving through a gel matrix than the same piece that is coiled upon itself. An analogy would be to equate this situation to your classroom in which all the desks have been randomly pushed together. An individual student can wind his/her way through the chair maze quickly and with little difficulty, whereas a string of students would require more time and have difficulty working their way through the maze.
The restriction enzymes used in this investigation are EcoR1, BamH1 and HindIII. You will use gel electrophoresis to separate the resulting DNA pieces. To help see the pieces, you will stain the gel with a chemical that will combine with the DNA causing it to take on a blue color. A permanent record of the gel can be made by photographing it with a Polaroid camera or photocopying it on a photocopy machine.
* The student will identify restriction enzymes and their specificity.
* The student will determine the number of restriction sites on Lambda DNA.
* The student will visualize DNA pieces within a gel and effectively communicate this visualization.
* The student will estimate the size of each DNA piece cut by a specific enzyme.
1. Lambda DNA 10. Power supply
2. (0.8%)Agarose 11. Loading dye
3. 1XTBE buffer 12. 4 Microcentrifuge tubes
4. Micropipet (1-20 ul) 13. Micropipet tips
5. Gel tray, comb and box 14. Distilled water
6. Microcentrifuge tube holder 15. Permanent marker
7. Masking tape 16. Semi-log graph paper
8. Enzymes-BamH1, EcoR1, 17. Millimeter ruler
Hind III 18. Uncut Lambda DNA
9. 2X Restriction buffer
Part A: Restriction of Lambda
1. Label 4 microcentrifuge tubes as follows:
H=HindIII (-)=No enzyme
2. Use and mark the following table with checks as you add chemicals to each tube. Read down each column, adding the same chemical to all tubes. You only need one tip per chemical if you don't contaminate it by touching any other chemical in the tube. You can avoid contamination by placing each new chemical at a different spot on the side of the tube. Use a fresh tip for each different chemical. Remember to keep the enzyme tubes on ice and to add the enzyme last!!!
Tube Lambda Buffer BamH1 EcoR1 HindIII Water B 5 ul 6 ul 1 ul E 5 ul 6 ul 1 ul H 5 ul 6 ul 1 ul (-) 5 ul 6 ul 1 ul
3. Seal each tube and place it into the microcentrifuge. Be sure to balance the centrifuge by spacing the tubes evenly inside. Pulse spin the centrifuge (push button for a few seconds) to make sure that the contents are mixed. Place the tubes in the microcentrifuge rack and incubate at 37o C (either in a water bath or incubator) for at least two hours or leave them on the lab counter over night.
Part B: Preparing an Agarose Gel
1. Prepare a gel tray by taping the ends with masking tape as instructed by your teacher.
2. Place the comb near one end of the tray (approximately of an inch) and pour melted agarose into the tray. (The agarose in this activity has a concentration of 0.8%)
Pour in just enough melted agarose to cover of the height of the comb teeth. Do Not move or handle the gel tray until the gel has solidified (about 10 minutes or until it appears cloudy).
Part C: Separation by Electrophoresis
1. Gently remove the gel comb by lifting it straight up and out of the gel.
2. Remove the tape from the gel tray and place the tray with the gel into the gel box. Be sure to place the gel so that the wells are closest to the negative (black) electrode.
3. Fill the gel box with enough 1X TBE buffer to barely cover the gel.
4. Dial the micropipet to 2 ul and add this amount of loading dye to each tube from the restriction digest in the above procedure. If you place the drop of dye along the side of the tube being careful not to touch the tube's contents with the pipet tip, you can load all tubes with the same tip. Place the tubes in the microcentrifuge and pulse spin for a few seconds. Remember to balance the centrifuge by spacing the tubes evenly inside the centrifuge.
5. Dial the micropipet to 14 ul and load the gel according to the following scheme. Be sure to change tips between EACH tube.
Lane Tube 1 H 2 B 3 E 4 (-)
6. Place the lid on the gel box (remember black to black and red to red). Plug the gel box wires into the power supply (again black to black and red to red). Turn on the power supply and set it at the voltage specified by your teacher. Let the gel run for approximately 90 minutes.
Part D: Staining and Imaging DNA
1. Mark a staining tray with your initials and period.
2. Carefully place your gel into this tray by sliding it off the end of the gel tray.
3. Now add enough DNA stain to just cover the gel.
4. Allow the gel to stain for 30 minutes.
5. To destain, pour the excess stain back into the stain bottle and rinse off your gel several times with water.
6. Then fill your staining tray with enough water to cover the gel.
7. Let stand for 1 1/2 hours and then pour off the excess water.
8. Place the staining tray and gel in a zip lock bag and seal it. You can store the gel for several days at room temperature for future analysis.
1. Label the lanes according to the table above in section C4.
2. Label the positive and negative ends of the gel ( ).
3. Place your gel upon the light source provided and record the result on the diagram.
( ) ( )
4. Linear DNA fragments migrate at rates inversely proportional to the log10 of their base pair length. The table below gives the base pair sizes of the different DNA pieces from the HindIII restriction digest of the lambda virus. Measure the distance in millimeters from the bottom of the well to the bottom of each band in the HindIII digest and place that measurement next to the base pair size that it matches.
HINDIII ECORI BAMHI
Band # Distance Base Pair Distance Estimated Distance Estimated (mm) Size (mm) Base Pair (mm) Base Pair
1 23,130 2 9416 3 6557 4 4361 5 2322 6 2027
5. By using the known base pair sizes of the bands in the HindIII digest as reference points we can estimate the other band sizes. The term given to these reference points is a ladder.
6. We will use the ladder to estimate the base pair sizes of the EcoRI digest and the BamHI digest.
7. On semi-log graph paper, mark the distance (mm) on the x-axis (horizontal axis) and the base pair size on the y-axis (vertical axis). Plot and connect these points in a best-fit straight line.
8. Measure the distances migrated for each of the bands in the other two digests. Remember you need to be consistent: measure from the bottom of the well to the bottom of the band. Record each measured distance in the appropriate box in the data table.
9. Refer to the graph by moving along the x-axis to the designated distance; then move up until you reach your best-fit line. From this point on the best-fit line, find the corresponding height on the base pair size axis (y-axis). Read the size measurement indicated on the base pair size axis. Record the measurement in the appropriate box.
1. What do the bands on the gel diagram represent?
2. What is the connection between the restriction enzymes and these bands?
3. How many bands are in lane 4? What does this band represent?
4. Compare lane 4 with the other lanes. Do any of the other lanes have a band in the same position as lane 4? Why or why not?
5. Which lane has the smallest piece of DNA? How do you know?
6.a. How many pieces did HindIII produce by cutting Lambda DNA?
b. How many times did this enzyme cut the DNA?
7.a. How many pieces of DNA did the BamHI restriction digest produce?
b. How many times did this enzyme cut the DNA?
8.a. How many pieces of DNA did the HindIII restriction digest produce?
b. How many times did this enzyme cut the DNA?
9. To which electrode did the pieces of DNA move? Why?
Remember that the three samples of DNA started out the same. Assuming that each sample was cut into pieces by the addition of three different restriction enzymes, how does a restriction digest give evidence that each enzyme cuts the DNA at different locations?
A plasmid is a small circular piece of DNA. Plasmids have restriction sites and usually carry a specific gene - often an antibiotic resistance gene. Since they are small, one restriction enzyme will usually only find one site for its specific cutting capabilities. The name of a plasmid contains the gene which it carries preceded by a "p": for example, pAMP is the name for the plasmid that contains the gene for ampicillin resistance. Following is the plasmid map for pAMP. Notice that the map contains the location of the gene, the position of the origin (which is the spot at which replication begins), the names of the restriction enzymes, and their restriction sites designated by the base pair distance. Therefore, from a plasmid map a research scientist can determine which enzymes can most effectively be used to cut out the gene in order to transfer that gene to another plasmid. The scientist must be careful that ALL of the gene is removed but does not want unnecessary amounts of plasmid DNA.
After viewing the plasmid, determine which enzymes would be best for cutting the ampicillin gene out of pAMP. In addition, determine the size in number of base pairs of the cut piece of plasmid.
A research scientist will often want to combine the genes from two plasmids to make a new organism that contains the resistance to two antibiotics. When the scientist proceeds with this technique, the new organism must contain the complete genes and an origin in order for replication to occur.
The plasmid maps for two different plasmids follow: pAMP and pLacZ. Select the restriction enzymes that a scientist would use to create the pAMP/LacZ.
Draw all possible plasmid combinations that could recombine including pAMP/LacZ and name them. Determine their sizes.