Student Instructions for Restriction Digest (NEB)

DNA Restriction Enzyme Mapping1

S2501, Contemporary Biology Laboratory, Columbia

Summer 2009

1Adapted from D. A. Micklos and G. A. Freyer, DNA Science, a First Course in Recombinant DNA Technology, Cold Spring Harbor Laboratory PressCold Spring Harbor, N.Y., 1990, ch. 3, and pp. 247-275.

 

I. Introduction

Molecular biology is a hybrid discipline that arose from a confluence of disciplines: genetics, physical chemistry, X-ray crystallography, biochemistry, microbiology, bacteriology, and virology. It represents the antithesis of the notion that physics, chemistry, and biology are separate and unrelated divisions of the natural sciences.In the beginning of the 20th century, physics and chemistry were united by quantum theory, which explored the fine structure of matter and its associated energy. At that time, biology was a field concerned with the direct observation and description of complex natural phenomena. Then, beginning in the 1920s and 30s, ideas derived from quantum mechanics began to infuse biology. It began to be understood that the behavior of any complex biological system, whether it is a bacterium, a plant, a frog, a leopard, or a human being, is based on the physical and chemical behavior of elementary particles. Molecular biology borrowed from the physical sciences the rigorous use of model systems—simplified abstractions of reality in which variables could be limited and experimental situations could be controlled. The development of molecular biology was in large part driven by the quest to find increasingly purer and more powerful abstractions of essential biological processes. Experimental systems using complex, multicellular organisms (such as pea plants and fruit flies used in the early 1900s) began to be replaced first by simple, one-cell organisms (bacteria and their attendant viruses, beginning in the 1940s) and then by purified cellular components (such as DNA and proteins, beginning in the 1960s).

II. Tools

A. Restriction Enzymes

Restriction enzymes were first discovered in the 1950s when evidence for a sort of primi-

tive immune system was found to exist in bacteria. It was observed that certain strains of E. Coli were resistant to infection by various bacteriophages (viruses that infect bacteria). This phenomenon seemed to be a property of the bacterial cell itself, an ability to restrict the growth and replication of the attacking phages. In 1962, at the University of Geneva, it was found that the resistant bacterium possessed an enzyme system that selectively recognized and destroyed foreign phage DNA within the bacterial membrane and at the same time modified the chromosomal DNA of the bacterium to prevent self-destruction.  Several years later, at Harvard University, extracts of E. coli were made which were found to efficiently cleave phage DNA. These extracts contained the first known restriction endonucleases, enzymes that attack and digest internal regions of the DNA of an invading bacteriophage but not that of the host. This was shown to occur because, in addition to a restriction (cutting) activity, these enzymes also possessed a modification (protecting) activity. However, because the cutting activity of these enzymes was not specific, i.e., the enzymes cut at random places within the phage DNA, they were of no practical value as tools for manipulating DNA. Then, in 1970, at Johns Hopkins University, a new kind of restriction enzyme was found, one that could cut at a specific site within the phage DNA, while at the same time protecting the same site on the bacterial host’s DNA. The protection was accomplished by adding a methyl group to the host’s site, thus disguising it. This new enzyme was isolated from Haemophilus influenzae, and was called HindII. It cleaved DNA predictably, cutting within a specific sequence of base pairs, regardless of whether the DNA was from a small virus that infects monkeys (Simian virus 40 (SV40)) or a virus that infects E. coli. The resulting restriction fragments could then be separated by size in an electrical field. The order of the fragments (and corresponding restriction sites) could be deduced in the 5,000 nucleotide circular chromosome to create a restriction enzyme map that was then related to the existing genetic map of SV40.

B. Agarose Gel Electrophoresis

Gel electrophoresis takes advantage of the fact that, as an organic acid, DNA can be

negatively charged, depending on the alkalinity of the surrounding buffer. DNA owes its acidity to phosphate groups that alternate with deoxyribose to form the rails of the double helix ladder. In a slightly basic solution, negatively charged oxygens radiate from phosphates on the outside of the DNA molecules. When placed in an electric field, DNA molecules are attracted toward the positive pole (anode) and repelled from the negative pole (cathode). During electrophoresis, DNA fragments sort by size in the gel. The porous gel matrix acts as a molecular sieve through which smaller molecules can move more easily than large ones, and the distance moved by a DNA fragment is inversely proportional to its molecular weight. In a given period of time, smaller restriction fragments migrate relatively far from the origin compared with larger fragments. DNA fragments in different size ranges can be separated by adjusting the agarose concentration: low agarose concentrations produce a loose gel that separates large fragments, whereas a high concentration produces a stiff gel that resolves small fragments. In the gel we use in this experiment, we chose an agarose matrix that can efficiently separate DNA fragments ranging in size from about 100 nucleotides to about 5,000 nucleotides. To do this, we use a 1.2 % gel, which turns out experimentally to be the size of molecular sieve that will effectively sort, and resolve, the fragments generated by our digestions.

III. Experimental Objective

This experiment involves mapping the number and relative positions of cutting sites for

three restriction enzymes, EcoR1, HincII, and PvuII, on circular plasmid pBR322, the first artificially produced plasmid to be used routinely in recombinant DNA experiments. This plasmid was originally derived from the bacteria E. coli and was constructed to have two antibiotic resistance genes, tetracycline and ampicillin. Restriction enzyme mapping is important because it is the first step in characterizing a DNA fragment that has been ligated into a vector (usually a plasmid), providing necessary data for applications such as DNA sequencing, gene localization, and site-directed mutagenesis.

IV. Notes on Experimental Procedure

Since molecular biology deals with small quantities, be prepared to measure liquid quantities on the order of microliters (a millionth of a liter) (in general, wear gloves for work in molecular biology). A few notes on the use of the micropipettor may be helpful:

1. Rotate the volume adjuster to the desired setting, which is shown in a window on the side of the micropipettor. The line between two of the digits corresponds to a decimal point.

2. Place the appropriate tip on the end of the micropipettor. The p10s (those pipettes that can pull up and expell a total volume of 10 μls use a very small diameter tip compared with a p20

or above. Use a new tip for each new solution.

3. When withdrawing or expelling fluid from a microfuge tube, always hold the tube firmly between thumb and forefinger. Hold the tube at nearly eye level to observe the change in the fluid level in the pipette tip.

4. Each tube must be held in the hand during each manipulation. Grasping the tube body,

rather than the lid, provides more control and avoids contamination.

5. Hold the pipettor almost vertical when filling.

6. Most micropipettors have a two-position plunger with “friction stops.” Depressing to the first stop selects the desired volume to be withdrawn from the tube. Depressing to the second stop introduces an additional volume of air to blow out any solution remaining in the tip. Pay attention to these friction stops, which can be felt with the thumb.

7. To withdraw a sample from the reagent tube:

a. Depress the plunger to the first stop and hold it in this position while you dip the tip

into the solution to be pipetted and gradually release the plunger. You should see liquid being drawn into the tip.

b. Slide the pipette tip out along the inside wall of the reagent tube to dislodge excess

droplets that may be adhering to the outside of the tip.

c. Check that there is no air space at the very end of the tip. To avoid pipetting errors,

learn to recognize the approximate level that particular volumes fill the pipette tip.

8. To expel a sample into the reaction tube:

a. Put the pipette tip into the reaction tube into which you wish to empty the sample,

near the bottom, touching the tip to the inside wall of the tube. This will allow a capillary effect

to help draw the fluid out of the tip.

b. Slowly depress the plunger to the first stop to expel the sample. Now depress the

plunger to the second stop to blow out the last bit of fluid.

c. Holding the plunger in the depressed position, slide the pipette out of the reaction

tube to avoid sucking any liquid back into the tip.

d. Eject the tip into a waste beaker near you on the lab bench.

9. To prevent cross contamination of reagents:

a. Add the appropriate amounts of a single reagent sequentially to all reaction tubes.

10. Then, using a new tip, add the next reagent, and so on.

b. Release each reagent drop onto a new location on the inside wall, near the bottom of

the reaction tube, being careful not to touch any drops already added to the tube. The same tip

can be used to pipette the same reagent into each reaction tube.

c. Use a fresh tip for each new reagent to be pipetted.

d. If a tip becomes contaminated, switch to a new one.

V. Mapping pBR322

A. Setting Up the Restriction Digests

1. Label six 1.5-ml microcentrifuge tubes, as shown in the matrix below. The restriction enzyme digests will take place in these tubes.

2. Use the matrix below as a checklist while adding the reagents to each reaction tube. Read down each column, adding the same reagent to each appropriate tube (all these reagents are in either in the ice buckets on your bench or in stock tubes at the front of the lab). Add the reagents in the order described in the steps that follow the matrix.

 
Student Set-ups:

Digest Setups

Tube #

pBR322-DNA

Buffer # and amount (10X)

Enzyme

(1 µL of each enzyme)

BSA 

(10X) 

dH2O

1

3 µL

EcoRI Buffer/ 1 µL

EcoRI

 1 µL

 4 µL

2

3 µL

Buffer 3/ 1 µL

HincII

 1 µL

 4 µL

3

3 µL

Buffer 3/ 1 µL

PvuII

 1 µL

 4 µL

4.

3 µL

Buffer 3/ 1 µL

PvuII, HincII

 1 µL

 3 µL

5.

3 µL

Buffer 3/ 1 µL

BamHI, PvuII

 1 µL

 3 µL

6.

3 µL

Buffer 3/ 1 µL

EcoRI, HincII, PvuII

 1 µL

 2 µL

 

3. Add 3 μl of pBR322 to the reaction tubes.

4. Use a fresh tip to add BSA (bovine serum albumin- helps the enzymes work better)..

5. Use a fresh tip to add the appropriate volume of distilled water to each reaction tube (the  amount needed will bring the total volume of the reaction to 10 μl.

6. Use a fresh tip to add 1 μl of 10X buffer (only the EcoRI uses a different buffer) to each reaction tube.

7. Use a fresh tip to add each different restriction enzyme to the appropriate reaction tube.

8. After carefully adding the required reagents to each tube, close the tops of the tubes and microfuge them briefly in the microfuge on your bench in order to bring all the reagents to the bottom of the tube. (The reagents need to be near each other in order to react.)

9. Place all of the reaction tubes in a “floater” and place the floater in the 37oC water bath. Incubate for 2 hours..

B. Casting a 1.2% Agarose Gel

1. Let the agarose cool to a temperature that will allow you to touch the flask (this will be about 60°C). While the agarose is cooling, make your gel mold liquid-tight by putting masking tape on either end are pressed against the sides of the box. Insert the thick side of the comb into the slot at the top of the mold. You will be creating 8 wells.

2. Pour your gel into the gel mold and let it set. If you see any bubbles on the surface of the gel after pouring, scoot them to the edges with a pipette tip.

Incubate for ~2 hours @ 37C.  Then refrigerate or freeze until ready to load gel.

 

Next Day:

6. Remove your gel from the baggie.  Carefully remove the making tape.

7. Pour running buffer (on your benches in large bottles) into the gel box so that it covers the gel by about a few millimeters (make sure the well edges are submerged).

C. Load Gel and Electrophorese

1. Retrieve your tubes from the front table.

2. Add 2.5 μl of loading dye to each reaction tube. The loading dye is a dense solution containingvarious pigments. The solvent here is a dense sucrose solution that should allow the colored solution to sink into the wells. The blue and purple pigments will tell us when to stop the current since one of the pigments travels at about the same rate as the smallest fragment of the DNA does.

3. After adding the dye, close the tops of the tubes, and microfuge.

4.. . Load 12.5 μl from each reaction tube (the total volume in the tube) into a separate well in the gel, beginning with the second well. Note that you will now need the p20 micropipette in order to accommodate the total volume of 12.5 μl. Make a note for yourself about which digest is in which well. Load from the left, proceeding in order through the last digest from the tube (see chart below)NoteUse a new  tip for each sample. Be careful not to push the tip of the pipette through the bottom of the gel.

5. Connect the leads to the power source and turn it on. Red indicates the positive end and black indicates the negative. Remember that since DNA is negative with the buffer we are using, you will want to position the leads so that the DNA is running toward the positive end.

7. Electrophorese until the leading dye has run down the gel to between the numbers 4 and 5 which appear on the side of the gel. Then turn the current off, or if another gel is still running on your power source, disconnect the electrodes for your gel. (200V for 20 minutes).

D. View and Photograph

1. Wearing gloves, take out the gel mold with the gel in it and slide the gel into staining tray onwhich you have written the table and period number (on the edge of the staining tray).

2. Pour enough Fast-Blast Stain (1X) to cover the gel and put on the rotating table.

 

Loading Pattern for Gel

Use 2.5 µl of 6X Blue Gel Loading Dye for the 10 µl reaction. Mix well before loading gel.  Load all the sample on the gel



 

Lane contents

 

Band sizes (bp)

 

Lane 1

 

HindIII Digest Control

 



 

Lane 2

 

EcoRI

 



 

Lane 3

 

HincII

 



 

Lane 4

 

PvuII

 



 

Lane 5

 

PvuII, HincII

 



 

Lane 6

 

BamHI, PvuII

 



 

Lane 7

 

EcoRI, HincII, PvuII

 



 

Empty

 

 



 

Bio-Rad: 0.25X TAE; 200 V, 20 minutes
Stain overnight in FastBlast Blue stain 1X


Next Day:

  1. View gel, measure distances of bands from the wells, record in data table.
  2. HindIII (known base pair sizes starting from the well; 23,130, 9416, 6557, 4361, 2322, 2027, 564.
  3. Use a semi-log graph to generate a standard curve for HindIII control (x-axis- distance band migrated (mm); y-axis (semi-log from 100 to 100,000).
  4. Determine sizes of unknown bands.
  5. 1. Draw a simple pBR322 restriction map showing the sites for enzymes used, in relative locations;

    2. Predict the expected number of linear DNA bands following complete digestion with each enzyme;

    3. Predict the expected sizes of those bands and use the size marker to help identify them on your gel;

    4. Explain what you see when pBR322 is digested with HincII;

    5. Explain why HincII  is expected to have many more restriction sites in pBR322 than the otherenzymes;

    6. Comment on any unexpected bands in your gel.

    (questions from http://www.csus.edu/indiv/r/rogersa/protpBR322.pdf )

     

 

 

DNA MAPPING: Post-Lab Questions to consider for your discussion.

1. What are two functions of the loading dye?

2. How does Fast-Blast visible dye stain DNA?

3. To cast the electrophoresis gels, why did we use buffer to dissolve the agarose; why not just use water? Please suggest 2 reasons.

4. To run our digests in the electrophoresis chamber, why did we use a buffer? Why not just use water?

5. In order to run our electrophoresis chambers, why did we cover the gels with just a little buffer; why not use quite a lot?

6. What would happen if we ran the gel with the electrodes reversed?

7. What might account for “smearing” (i.e., no discrete bands, just one big blurry smear)?

8. Why might various faint bands appear in some of the lanes?

9. How would decide whether these faint bands were “real” bands or just artifacts?

10. Restriction enzymes are generally named for the organism from which they were derived. From what organism was PvuII derived?

 

Rubric:  (23)

Title (1)

Data Table (4)

HindIII Standard graph with notations to show how you determined unknown band sizes in basepairs. (4) (some helpful info...for a resource, don’t email the other teacher whose site I’ve linked  http://www.occc.edu/bbdiscovery/documents/MWfromgel.htm )

 

Possible Restriction Maps (at least two) to show possible order of fragments on pBR322 BASED ON YOUR DATA ONLY. (8) 

Discussion (background info and compare to the actual pBR322 plasmid map(4)

References (2)