1 Bio 121 LAB 11 INSTRUCTIONS - DNA II In the first part of today's lab we will demonstrate that the DNA which we extracted last week can create heritable changes in the phenotype of bacterial cells. We will transform this plasmid, which carries a gene encoding a protein which destroys the antibiotic ampicillin into bacterial cells which are sensitive to ampicillin. We will then verify that the cells which grow on ampicillin indeed contain this plasmid by testing for the presence of the green fluorescent protein which is encoded by another gene carried on this plasmid. In the second part of today s lab we will use restriction enzymes to analyze the DNA which we extracted last week. In the third part we will extract and fingerprint our own DNA, using the same approach that the FBI uses for forensic analysis Genetic transformation Genetic transformation entails transporting DNA molecules through the cell wall and across the plasma membrane into the cytoplasm. It is therefore necessary to make the cells "competent" to take up DNA from their environment. To do so you will suspend cells of E. coli XL1-Blue in ice-cold 50 mm Ca 2+ for 15. It is not entirely clear what "competence induction" does, but it is thought to loosen up the cell wall so that circular DNA molecules can be slipped through, while maintaining the cells' viability. In any case, when cells treated in this way are mixed with DNA and then subjected to a heat shock, a few will take up the foreign DNA into the cytoplasm and begin to make the proteins encoded by the new plasmid. Therefore, after a brief recovery period to give cells time to make the proteins which destroy ampicillin, cells which took up the plasmid should be able to grow on medium containing ampicillin whereas those which did not will be killed by ampicillin. Consequently, if the plasmids contain origins of replication which tell the cells how to replicate it and how many copies to make, colonies of bacteria all descended from a single cell which took up a plasmid should form at the site on the plate where this cell was deposited. Each cell in the colony will contain one or more copies of the drug-resistance plasmid. DNA analysis by digestion with restriction enzymes Superficially, all DNA looks similar, since the differences between DNA molecules are in the specific sequence of bases. Therefore, the most definitive way to analyze a DNA molecule is to determine its nucleotide sequence. However, sequencing DNA is costly, time-consuming, and technically difficult. Consequently, researchers usually start by analyzing the molecule with proteins called restriction enzymes, which cut DNA at specific sequences called "restriction sites." Each restriction enzyme recognizes and cuts a specific sequence, therefore, we can determine the number of times that the sequence recognized by a particular enzyme occurs within a DNA molecule by measuring the number of fragments which it creates (fragments created by restriction enzymes are called "restriction
2 Instructions for Bio 121 lab 11: DNA II page 2 fragments"). We can also determine the distance between restriction sites by measuring the size of each restriction fragment using gel electrophoresis. We can also map the relative positions of restriction sites for different enzymes by measuring the size of the fragments created when the molecule is digested with both enzymes. Consider the following example of a circular molecule of 5,400 base pairs which has two recognition sites for the enzyme HindIII and one for the enzyme Eco R I. Eco RI creates a single fragment 5,400 bp long, Hind III creates two, 1000 and 4400 bp, respectively, while a double digest creates three, 300, 700 and 4400, respectively. Eco RI thus cuts within the Hind III fragment, 300 bp from an end, but it would require further mapping to determine which end it cuts close to. Figure 2: map of pglo (5,400 basepairs). If we were to separate the fragments created by digesting pglo with Eco RI, Hind III or Eco RI and Hind III by gel electrophoresis we would expect to see the following pattern.
3 Instructions for Bio 121 lab 11: DNA II page 3. Thus, by digesting a DNA molecule with a number of restriction enzymes either individually or in pairs and then measuring the length of the resulting fragments a researcher can determine the distance from one restriction site to another to create what is called a "restriction map." Restriction maps provide a distinct "fingerprint" for a particular molecule or region of a molecule, which we will see later in the course can be used to identify the source of particular DNA molecules. Fingerprinting human DNA: Each human has a unique DNA sequence; this is what makes us unique. Even identical twins have acquired some differences in their DNA by the time they are born. These differences allow us to distinguish one human from another based on their DNA sequence. Many DNA sequences, called DNA polymorphisms, have been identified that are highly variable between individuals. These are useful for human genetics because they are codominant since we will see both alleles in heterozygous individuals. They are also useful for identifying individuals. The FBI has found that it can determine a unique "fingerprint" for every individual using thirteen of these polymorphic sequences. These form the core of the Combined DNA Index System (CODIS) database ( ).
4 Instructions for Bio 121 lab 11: DNA II page 4 Today we will use PCR to determine which alleles we each have for DNA polymorphisms found on chromosomes 4 and 16. The polymorphism on chromosome 4 is one of the core loci used by the FBI. It is located in the third intron of the human alpha fibrinogen gene, and we have 12 to 51 copies of a 4 base repeat at this location ( The other polymorphism is due to a 300 bp Alu sequence: some of us have it, and some of us don't. Neither of these polymorphisms appear to affect us in any way, but we can detect their presence by PCR. When we amplify these regions by PCR using primers which bind on either side of the Alu sequence on chromosome 16 we form bands 550 bp long in individuals who lack this sequence and 850 bp long in individuals who have it, while on Chromosome 4 we generate fragments ranging from 158 to 314 bp long. Individuals who are homozygous for either allele will form single bands, while heterozygotes will form two bands. We can thus determine the frequency of each allele in every lab group by running PCR on our DNA and separating the products on a gel. This week we will extract our DNA and run the PCR reactions. Next week we will analyze the products by gel electrophoresis. Experimental Procedure Experimental procedures in molecular biology frequently require substantial waiting periods between one step and the next. For example, you need to allow the cells to recover for 30 minutes after taking up DNA in order to give them time to make the protein which destroys ampicillin before plating them on medium containing ampicillin. You will therefore be doing several things at once today, and will need to keep track of time. You will first begin the bacterial transformation. Then you will pour the gel, load and run your DNA samples while you finish the transformation procedure. While the gels are running you will also extract some of your own DNA, and fingerprint it using microsatellites. 1) DNA analysis by digestion with restriction enzymes You will analyze your DNA by digesting it with the restriction enzymes Ase1 and Bsp E1 and both enzymes at once. You will then determine the size of the resulting restriction fragments by gel electrophoresis and comparing the distances migrated with the distance migrated by standards of known size, just as you did last week. You will also load a lane with undigested DNA to confirm that your restriction enzymes worked 1. Obtain four 1.5 ml microcentrifuge tubes and label them with your icon, then number them Add 10 µl of the pglo plasmid DNA which you prepared last week to each using a micropipettor. 3. a. Add 10 µl " Eco 0109i" to tube 1, using a new pipet tip! DO NOT CROSS- CONTAMINATE THE RESTRICTION ENZYMES OR YOU WILL BE CONDEMNED TO 3 WEEKS FILLING PIPET TIP BOXES! b. Add 10 µl " Nco I " to tube 2. c. Add 10 µl " Eco 0109i + Nco I " to tube 3. d. Add 10 µl "TE" to tube Place the tubes in the 37 C water bath and leave them for at least 30 minutes. 5. After 30 minutes put on a pair of gloves and keep them on for the rest of the lab! The loading dye contains the fluorescent dye EZ-Vision to enable us to see and photograph the DNA. Since it binds DNA, it may be carcinogenic. 6. Add 4 µl "loading dye" to each tube, then load your gel and run it as you did last week.
5 Instructions for Bio 121 lab 11: DNA II page 5 7. a. Load 10 µl from tube 1 into the outside lane (lane 1) using a micropipettor. b. Load 10 µl from tube 2 in lane 2 c. Load 10 µl from tube 3 in lane 3 d. Load 10 µl from tube 4 in lane 4 e Load 10 µl of size standards in lane 5 8. Attach the electrophoresis chamber to the power supply and run until the blue dye band from the top row of samples is 2/3 of the way down the gel. For our system, this usually takes 60 min. After electrophoresis is completed, turn off the power supply and disconnect the electrophoresis chamber. 9. Your instructor or T.A. will photograph the gel for you. You should observe several bands in lane 4. This is because some of your plasmids will be uncut, some will be broken during extraction, and some of the uncut plasmids are joined together to form dimers and trimers. Uncut plasmids travel faster than broken plasmids because they are coiled into a more compact shape, whereas dimers and trimers move more slowly because they are larger. After photographing the gel dispose of it into the jar provided. 10. Graph the length of your size standards vs. the distance they traveled on the graph on page 4 of your datasheet. Your size markers are bacteriophage lambda digested with Eco RI and Hin diii. The fragments are 120, 564, 831, 947, 1375, 1709, 1904, 2027, 3530, 4268, 4973, 5148 and bp, respectively, although you will rarely see the 120. Note that the 1904 and 2027 bp fragments frequently run together, as do the 4973 and 5148 bp fragments. 11. Report your findings on your datasheet. Your introduction should explain what a restriction enzyme is, why it is useful for analyzing DNA molecules, and the specific purpose of this experiment. Your results should include sentences describing the purpose of the experiment, where the results are, and highlighting the key results. They should also include a photograph of your gel, and estimates for the sizes of each restriction fragment observed in lanes 1, 2 and 3. In your discussion you should explain why you obtained the results observed in lanes 1, 2, 3 and 4. 2) Bacterial transformation You will transform one aliquot of cells with the plasmid pglo, and a second aliquot of cells with buffer without DNA to confirm that the competent cells are ampicillin-sensitive until they take up a plasmid conferring ampicillin resistance. The transformed cells from both suspensions will be plated onto a growth medium called LB containing 100 µg/ml ampicillin (LB Amp). To provide a solid surface for them to grow on the medium has been solidified with agar, a complex mixture of carbohydrates isolated from seaweed which does not have any nutritional value. Only bacteria carrying pglo should grow on these plates. To confirm that they survived the procedure you will also plate aliquots from each tube on LB medium without ampicillin, and to check that the ampicillin-resistant cells indeed contain pglo you will see whether the colonies fluoresce green when illuminated with a UV lamp. 1. Obtain two sterile 1.5 ml microcentrifuge tubes. Label one tube "+" and one tube "-". 2. Pipette 0.25 ml of sterile 50 mm CaCl 2 into each of the culture tubes. Keep on ice. 3. Scrape a loop-full of E.coli off a master plate and disperse in the + tube. Repeat for the - tube then place both tubes on ice.
6 Instructions for Bio 121 lab 11: DNA II page 6 4. To the tube labeled "+", add 10 µl of a solution containing the DNA which you extracted last week. Add nothing to the tube labeled "-". Incubate both tubes on ice for 15 min. 5. Carry the ice bath to the 42 C water bath and, holding the tubes by the tops, immerse them in the water bath for exactly 90 seconds, then return them to ice. (This is a "heat shock" that will promote absorption of any DNA bound to the cells.) 6. Add 250 µl SOC broth to each tube. 7. Leave each culture shaking at 37 C for at least 30 minutes to allow the cells to recover and start making the protein which destroys ampicillin. 8. Once the cells have recovered for at least 30 minutes plate the cell suspensions as follows: a. Spread 100 µl + cells on an LB AMP plate. using a glass spreader as demonstrated by your instructor. b. Spread 100 µl - cells on an LB AMP plate.. c. Spread 100 µl + cells on as LB (without ampicillin) plate d. Spread 100 µl - cells on as LB (without ampicillin) plate 9. After the plates have dried, invert them and incubate overnight at 37 C. 10. Next day, come back and enter the number of cells growing on each plate in Table III on page 5 of your data sheet. If there are more than 200, but individual colonies can be distinguished enter ">200." If individual colonies can not be distinguished enter "lawn." 11. Shine a UV lamp on your plates and record how many colonies glow green. 12. Report your findings on your datasheet. Your introduction should explain what a plasmid is, what genetic transformation is, why you expect a plasmid to confer antibiotic resistance to a host cell that takes it up, and the specific hypothesis of this experiment. Your results should include sentences describing the purpose of the experiment, where the results can be found, and the highlights of Table III. Your discussion should explain why you obtained more colonies on the LB amp plates with the cells transformed with "pglo" than with the cells transformed with "TE," and why you obtained more colonies on the plates without ampicillin than on the LB amp plates for both sets of cells. Fingerprinting human DNA with microsatellites You will extract DNA from your own cheek cells, then determine by PCR which alleles you have of two DNA polymorphisms. Next week you will analyze the products of these PCR reactions by gel electrophoresis. 1. Pour 10 mls 150 mm NaCl into a cup, slosh it around thoroughly inside your mouth, then spit it back into the cup. It should look pretty murky and gross; if not, try again. 2. Put 1.5 mls into a microcentrifuge tube and discard the rest into the booger beaker. 3. Spin the cells for one minute at 14,000 x g in a balanced microcentrifuge (centrifuge with a friend). 4. Discard the supernatant, then resuspend the cells in 30 µl extraction buffer. 5. Transfer to a fresh PCR tube containing 100 µl Chelex solution, and pipet in and out 10 times to mix.
7 Instructions for Bio 121 lab 11: DNA II page 7 6. Mark the lid with your icon, then give the tube to your instructor, who will first keep it at 58 C for 10 minutes, then at 99 C for 10 minutes. 7. Spin the cells for five minutes at 14,000 x g in a balanced microcentrifuge (centrifuge with a friend). 8. Transfer 3 µl of the supernatant to two 200 µl PCR tubes labeled with your icon and A or 2 on the lid. Be careful not to transfer any beads! 9. Bring your tubes to your instructor when you have done this. Your instructor will add 17 µl of Taq DNA polymerase stock (containing the buffer, MgCl2, dntps and appropriate primers) to your tubes, then will run 35 cycles of 94, 50, When finished we will store the tubes in the freezer until next week.