Exercise 3 - University of California, Berkeley



Exercise 3. Isolation of Plasmid DNA

Objectives

From this exercise, the structural differences between plasmid DNA and bacterial chromosomal DNA should be understood. These differences are used to separate plasmids from chromosomal DNA and other debris in a cell culture of E. coli. Various methods, both physical and chemical, are utilized throughout the plasmid isolation process. Students should realize the importance of each step as it pertains to the overall isolation scheme. The method of gel electrophoresis will also be introduced as a technique for separating DNA for visualization by staining with ethidium bromide.

PreLab Questions 2

Please limit your answers to a few sentences IN YOUR LAB NOTEBOOK

1. Under the conditions of this experiment, what happens chemically to cause the denaturation of chromosomal DNA? What phenomenon helps to prevent the denaturation of plasmid DNA?

2. What chemical detergent is used to precipitate out chromosomal DNA and high MW RNA? What effect(s) does this solution have in this experiment?

3. In this plasmid preparation procedure high and low MW RNA are precipitated out and discarded. Why then, do we still add RNase in the final suspension?

4. In what direction does the DNA run along the electrophoresis gel? Why?

5. Ethidium Bromide is a powerful mutagen (extremely carcinogenic) that may be fatal if inhaled and harmful if swallowed or absorbed through skin. It causes irritation to skin, eyes, and respiratory tract, and may cause heritable genetic damage. What precautions should you take while working with EtBr in this lab?

Background

Plasmids exist in bacterial cells as circular DNA molecules and can range in length from 1000 to 200,000 base pairs. In nature, plasmids evolved as harbingers of recombinant DNA. Some plasmids naturally carry genes that afford resistance to antibiotics and can provide a selective advantage to host cells. In a process called conjugation, plasmids can be physically passed from one cell to another, thus conferring the drug resistance to future generations. Several plasmids, such as pBR322 (an ancestor to pUC19), have been extensively characterized by molecular biologists. Plasmid pBR322 contains a gene for tetracycline resistance and a gene for ampicillin resistance. This information can be used very simply to separate cells that contain the plasmid from those that do not. Only cells containing the pBR322 plasmid will grow in a medium containing either of these antibiotics.

Plasmids are invaluable tools in genetic engineering. Well-characterized plasmids are applied as cloning vectors, or vehicles for the insertion of foreign DNA into a bacterial host. Entire genes or sets of genes can be spliced onto a plasmid, thus converting host cells into protein factories when the DNA blueprint is translated via cellular machinery. They are autonomous from the cell’s chromosomal DNA in that they can be replicated independently of the replication of genomic DNA. This replication can be controlled such that the copy number can be increased to over 1000 plasmids per cell. At this point the cell expends substantial energy into expressing the gene(s) of interest and producing the protein(s) desired by the genetic engineer.

Central to the task of plasmid manipulation is isolation from its bacterial source. Fortunately, plasmid DNA and cellular chromosomal DNA are physicochemically distinct enough to yield relatively simple procedures for plasmid separation. Conditions can be imposed which cause chromosomal DNA to be irreversibly denatured due to disruption of the hydrogen bonds involved in DNA base-pairing. Under these same conditions, the strands of circular plasmid DNA are tightly intertwined and are unable to separate from each other. This phenomenon, called supercoiling, can be illustrated with a simple demonstration. Suspend a shoe by its laces, then spin the shoe until it stops. Double helix “DNA” results. Now support the shoe, still holding the laces, and continue to twist the shoe. Tension in the laces causes the double helix to wind around itself, yielding “supercoiled DNA.” Supercoiling renders plasmid DNA far less susceptible to denaturation than the chromosomal type (Cantor and Schimmel).

Plasmids will be isolated from the host cells using a commercially available isolation kit (QIAGEN) that relies on the principles of the modified alkaline lysis method of Birnboim and Doly, and on the binding nature of DNA and a silica-gel membrane. Whole cells, derived from a single transformed colony, are pelleted in a centrifuge and re-suspended in a small amount of buffer. This buffer also contains ribonuclease A (RNase A), an enzyme that breaks down and separates contaminant RNA from the plasmid DNA. Then at high pH, and with detergent present, cell lysis is induced. A third, high salt solution is added to precipitate out the denatured chromosomal DNA and the insoluble salt of the detergent used, as well as remaining cell debris. This precipitate is pelleted out by centrifugation and discarded, while the supernatant is added to a column which contains a silica-gel membrane. The high salt condition induces the binding of DNA to the membrane while other materials present in the supernatant are washed through with centrifugation. The column is washed with a solution that contains a large amount of ethanol, to further remove unwanted materials. Finally, a low salt buffer is added to the column that induces the dissociation of the DNA and the silica-gel. One last centrifugation produces a solution of intact plasmid DNA in buffer.

DNA can be viewed and DNA fragment sizes can be deduced from gel electrophoresis. This invaluable technique separates fragments based on molecular weight. The DNA is loaded into wells at one end of the gel, and a charge gradient is imposed across the gel matrix. The DNA backbone consists of negatively charged phosphate groups, so all fragments migrate in response to the applied voltage. Mobility through the gel matrix is limited by the diffusivity of the DNA in the gel pores. Larger molecules have a harder time getting through the pores than do smaller ones, so small DNA fragments will move quickly through the gel while large ones stay behind. The molecular weight of each unknown fragment can be determined by running standard fragments of known size through the gel in an adjacent well. The gel is stained with ethidium bromide, a mutagenic chemical that binds to DNA, to visualize the fragments. Finally, the migration distance of each fragment is compared to the molecular weight standards, and sizes deduced. You will use this technique to simply visualize your isolated DNA in this exercise, and to determine several fragment sizes and orientations in the next exercise.

Procedures

Preparation of Suspension Culture (The TA will complete this portion the day before)

- you will do this yourself in later exercises

Sterile technique is required for this protocol. This is important to prevent the growth of foreign organisms; these will not contain the plasmid you are interested in isolating. Remember, to use a flame, cover agar plates, and restart contaminated specimens. Be careful not to contaminate agar plates with good colonies on them, as they will be limited.

1. Use a sterilized culture loop to pick a single colony from your plate of transformed E. coli. Dig in the agar around the colony. Manipulate the culture loop so that the isolated agar piece and the desired colony come free from the dish.

2. Inoculate the tube of LB/ampicillin medium (5 ml) by dropping in the agar cube that contains the bacterial colony. Cutting the agar out this way assures that you obtain a sufficient amount of cells to culture overnight.

3. Allow the culture to grow overnight in a 37°C shaker.

Plasmid Miniprep – Qiagen Spin Miniprep Plasmid Isolation Kit

(procedure taken from kit instruction manual)

Note: Sterile technique is not necessary for the rest of this exercise. (Why not?) Be sure to properly label all tubes so that they will not become confused with those of another group.

4. Transfer 2 1.5 ml of the overnight culture to a 2 1.5 ml microcentrifuge tube. Balance with another tube of water (or another group’s sample) for centrifugation. Centrifuge the tube for 30-60 seconds5 minutes at 1410,000 RPM in the microcentrifuge.

5. Discard the supernatant, transfer another 2 1.5 ml of overnight culture to the centrifuge tube, and centrifuge for 30-60 seconds5 minutes (you will pellet a total of 4 3 ml of culture).

4. Discard the supernatant and re-suspend the pelleted cells in 250 μl of buffer P1, the cell resuspension solution. (RNase A has already been added to this solution.) Use the vortexer at maximum speedmaximum speed to completely re-suspend the pellet.

5. Add 250 μl of buffer P2, the cell lysis solution and gently mix by inverting the tube 4-6 times. The solution should become viscous and slightly clear. DO NOT VORTEX the solution to mix as this can cause shearing of the DNA. Do not allow this reaction to proceed for more than 5 minutes or the plasmid DNA will begin to degrade.

6. Add 350 μl of buffer N3, the neutralization solution, and invert the tube immediately but gently 4-6 times to mix. Be sure that the solution is mixed thoroughly in order to avoid localized precipitation. (It should now be cloudy.)

7. Centrifuge in the microfuge at maximum speed13,000 for 10 minutes. Be sure to balance with water or another sample. While the sample is being centrifuged, prepare one QIAprep spin column.

8. After centrifugation of cell lysate, pipet the supernatant above the white precipitate and add to the spin column.

9. Centrifuge the column for 60 seconds. Discard the flow-through.

10. Wash the QIAprep spin column by adding 0.5 ml of buffer PB and centrifugeing for 60 seconds.

11. Wash the QIAprep spin column by adding 0.75 ml of buffer PE and centrifuging for 60 seconds.

12. Discard the flow-through and centrifuge for an additional 60 seconds to remove the residual wash buffer. This is very important! The column wash solution is mostly ethanol. Trace amounts of ethanol can inhibit reactions that you will be conducting in the following exercise.

13. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute the plasmid DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAprep spin column, let stand for at least 1 min, and centrifuge for 1 min.

14. The DNA is now in solution in the bottom of the tube. Discard the spin column. Close the tube and set it aside for the next section.

Preparing a 1.4 % Agarose Gel

(NOTE: The gel will be prepared for you for this exercise; however, you will need to prepare your own gel in Exercise 4, so you should familiarize yourself with this procedure.)

8. Mix up 700 ml total 1X TAE from a 50X TAE stock and Nanopure water. Prior to lab, calculate the volumes of stock and water.

9. Determine the amount of agarose you need to add to 70 50 ml of the 1X TAE buffer to obtain a 1.4 % solution (% means grams per 100 ml; a 1% solution would have 1 gram solute per 100 ml total solution). Mix the solution in a 250 ml Erlenmeyer flask; the agarose won't dissolve until the mixture is heated.

10. Stuff a Kimwipe loosely into the mouth of the flask. Heat the solution in the microwave for about 40 to 60 seconds. Try not to let it boil too hard. The Kimwipe is to prevent boiling over. Careful taking your flask out -- it's hot! Swirl the agarose solution around until you can't see any more beads of dissolving agarose.

11. Add 1 drop of ethidium bromide (EtBr) for staining. EtBr is a powerful mutagen and a known carcinogen! Wear double gloves, a lab coat, and glasses. Keep EtBr and all equipment it contacts in the designated counter space.

12. Allow the agarose to cool until it is comfortably warm (you can hold it against your wrist for a long time without experiencing pain). Gels poured too hot will warp the gel tray.

13. While allowing the agarose to cool, use laboratory tape to seal the edges of the gel tray. Arrange the tray and comb in the gel box for pouring the gel. The proper arrangement will be demonstrated. Orient the comb on one end of the plate so it rests ~1mm off the plate surface. This will allow the resulting wells to penetrate 2mm into the gel upon pouring.

14. Pour the agarose slowly into the center of the gel box and allow it to spread to the edges and among the teeth of the comb. Pour the entire volume you mixed up. The resulting gel layer should be about 3mm thick. Orient the comb on one end of the plate so it rests ~1mm off the plate surface. Check that there are no bubbles around the comb or in the middle of the gel. If you see any, move them to the edges with a pipet tip. Shift the comb around until it is bubble-free. When you're happy with the gel, leave it alone to harden.

Preparing Electrophoresis Samples

15. You will run two lanes of a DNA molecular weight standard and one lane of your isolated plasmid DNA on the gel. The molecular weight standard is bacteriophage lambda DNA which has been digested with a restriction endonuclease. (These will be discussed further in the next exercise.) Obtain two 0.5 ml microfuge tubes for preparing your samples.

16. Prepare a table which lists the lane number, name, and components of each sample that will be run on the gel (see below for components). The standards should be run on either side of the plasmid DNA sample.

17. A loading buffer must be added to the samples so that the DNA solution is denser than the electrophoresis buffer and will sink to the bottom of the gel well. The buffer also contains two dyes which migrate through the gel with different mobilities so that the approximate progress of the gel can be measured without actually visualizing the DNA. (The relative mobilities of the dyes are given in the Equipment and Reagents list.) The buffer is 6X concentrated; hence, you will add 1 5 μl loading buffer to a 6 30 μl total sample. Use 2 10 μl of your plasmid DNA and 1 5 μl for each of the two molecular weight standard lanes. Determine the amount of water you need to add to each sample to obtain 6 25 μl total volume (not including the loading buffer), and record the amount of each component you will use in your table. Now, prepare your samples according to this table. You may prepare the appropriate amount of sample for both standard lanes in the same tube; simply double the requirements for a single lane.

Loading the Gel

18. Once the gel has hardened (~35 minutes), gently rock the comb back and forth until free. Strip the tape from the wall of the mold and carefully place the gel in the horizontal apparatus.

19. Turn your gel around so the running direction is towards the positive (red) electrode. Why will the DNA run towards the positive electrode? Pour enough TAE electrophoresis buffer over your gel, being careful to get rid of trapped bubbles, so the buffer comes to about 1 mm above your gel (you can see this from the side of the gel box). You are now ready to load.

20. The technique for loading samples into a gel will be demonstrated. Draw the sample into the pipette tip. Position yourself so that you are stable and facing the gel box. Place elbows on the work surface and place your non-dominant hand against the shaft of the Pipetman to steady yourself. Lower the end of the tip into the well. Be careful not the puncture the bottom of the well and not to tear the sides. If the tip is not placed deep enough into the well, the sample will spill into the buffer. Slowly inject the sample. If the well has been loaded correctly it will look like a sharp blue square when viewed from the side. Do not take too long loading the gel, as this will allow the samples to disperse.

21. Check your gel once more for orientation (running direction). Plug the electrode leads into the power supply. Turn it on. Run for about 30 minutes to 1 hour at a constant 110 95 volts. Check the progress of the faint purple band to determine when to stop the gel. The gel should be stopped when the purple band is 1/2 of the way down. The current will gradually heat the buffer, and buffer overheating will warp the gel!!! Monitor the gel temperature, and consult the GSI if it begins to feel too warm (you may need to turn down the voltage at this stage).

Viewing the Gel

22. Turn off power supply, remove the top of the box, remove the gel tray.

23. The gel will be stained in a 0.5 μg/ml solution of ethidium bromide (EtBr). EtBr is a powerful mutagen and a known carcinogen! Wear double gloves, a lab coat, and glasses. Keep EtBr and all equipment it contacts in the designated counter space. Slide your gel from the tray into the staining solution. Do not touch the liquid surface with the gel tray, as it will become contaminated with EtBr. Soak for 20 minutes.

24. Viewing and photographing the stained gel will be demonstrated. Place plastic wrap on the surface of the UV transilluminator, and press it so that it is smooth. Using the scoop, remove the gel from the staining solution and slide it onto the transilluminator. Be careful not to splash touch EtBr on to the non-covered portions of the light table and counter. Turn the light on. Make sure to wear safety glasses, as UV light will damage your eyes. Do not look at the gel for more than a few seconds, or you will get “sunburned” by high intensity, carcinogenic UV radiation. Check you gel through the protective glass. DNA will appear as pink bands under UV light. Put the Polaroid camera over the table, take a picture of your gel, and develop it as per the instruction. This will also be demonstrated. Now, DNA will appear as bright bands against a dark gray background.

Guidelines for Analysis & Conclusions Section

(Remember, these are points you should consider and include in your analysis. This section, however, need not be limited to these specific guidelines.)

1. A traditionally used method for the isolation of plasmid DNA from bacterial cells (one that was commonly used prior to the availability of kits like the one we used) involves cell lysis followed by centrifugation of insoluble precipitates, just as it occurred in this exercise. However, the plasmid DNA is then isolated from the supernatant with a series of organic solvent extractions (e.g. phenol:chloroform) followed by precipitation with ethanol at low temperatures, and concluding with centrifugation at low temperature to obtain a pellet of nucleic acids that includes both DNA and RNA. The pellet is then dissolved in a low salt buffer that contains RNase A. Discuss the advantages of using the QIAGEN kit over this more traditional method.

2. The isolation method described above is easily scalable. That is, instead of harvesting 1.5 ml of cells, one could harvest 15 ml of cells and simply increase the amount of reagents used in each step by a factor of ten. This allows the isolation of a very large quantity of DNA. Does the QIAGEN kit appear to be easily amenable to scale-up? What would be the limiting factor for isolating DNA from a large volume of cells using the QIAGEN kit?

3. The lysis buffer (Buffer P2) contains a high concentration of sodium hydroxide (NaOH) and a detergent called SDS. What properties of SDS (sodium dodecyl sulfate) make it especially useful for use in a cell lysing solution? What is the mechanism of alkali cell lysis? Be specific.

4. At what point in the procedure is the chromosomal DNA denatured and how, chemically?

5. The DNA bands on an agarose gel migrate on a log scale, i.e. plotting the log of the molecular weight versus the distance traveled yields a linear relationship. Hence, the molecular weight standards can be used to make a calibration curve which can then be employed to determine the size of an unknown fragment. The sizes of the fragments in the standard are 12,000 base pairs (bp) through 2,000 bp in 1,000 bp increments, plus 1,650 bp, 1,000 bp, 500 bp, 400 bp, 300 bp, and below. (The low MW bands may not be clearly visible). Use this information to make a calibration curve and determine the apparent size of your isolated pUC19 plasmid DNA. Express the value you calculate in kilobase pairs (e.g. 9416 bp = 9.4 kbp), since your gel will not yield more accurate results than that.

6. Agarose gel electrophoresis is used to separate biological macromolecules based on differences in molecular weight, i.e. lower molecular weight components have a greater diffusivity through the gel matrix than heavier ones. The plasmid obtained from this experiment migrates much farther through the gel than one would expect, considering its molecular weight (.pUC19 is 2686 bps). (You must take my word for it on this one, since you don’t actually know what the true size of the plasmid is at this point!) PIf, prior to electrophoresis, the plasmid is can be treated with an enzyme that cleaves the DNA backbone;. T this results in a slower-moving band. Explain.

Equipment and Reagents

LB (Luria-Bertani) / Ampicillin Medium

1 liter deionized water

10 g bacto-tryptone

5 g bacto-yeast extract

10 g NaCl

pH adjusted to 7.0 with 5N NaOH; sterilized by autoclaving.

100 μg/ml ampicillin

QiaSpin Miniprep Kit

Sterile culture tubes

Microfuge

Sterile inoculating loops

1.5 ml microfuge tubes

0.5 ml microfuge tubes

Sterile pipet tips and Pipetman

Microcentrifuge tube rack

Electrophoresis unit; includes power supply, box, plate, and sample comb

UV light box

Molecular Weight Marker, Life Technologies (Gibco) 1 kb Ladder

Ethidium Bromide (EtBr), Stock Solution at 10 mg/ml (20,000X)

Agarose, electrophoresis-grade

DNA loading buffer for gels 10X

For 10 mL:

4 g sucrose

1. mL 10 mg/mL bromophenol blue

1. mL 10 mg/mL xylene cyanol

2 mL 0.5 M EDTA

filter sterilize through a 0.2 μm filter

50X TAE Gel Electrophoresis Buffer:

2 M TRIS 242 g/L

0.95 M Acetic Acid 57 g/L

0.05 M EDTA (as sodium salt, pH 8.0) 20.8 g/L

References

Bauer, W.R., Crick, F.H.C., and White, J.H., Supercoiled DNA, Sci. Am. 243(1): 118-133 (1980). [The topology of supercoiling.]

Birnboim, H.C., and Doly, J. (1979) Nucl. Acids Res. 7: 1513-1522.

Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, Part III, pp. 1265-1290, W.H. Freeman and Company, San Francisco.

Voet, D., Voet, J.G. (1990) Biochemistry, pp. 791-824, John Wiley and Sons, New York.

Sambrook, Fritsch, and Maniatis. (1989) Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.

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