LAB 7
DNA RESTRICTION for CLONING STUDENT GUIDE
GOALS
The goals of this lab are to provide the biotech student with experience in DNA digestion with restriction enzymes for the purpose of gene cloning, and to teach about the structure and function of plasmid DNA.
OBJECTIVES
After completion, the student should be able to:
1. Define what a DNA restriction endonuclease is and how it functions to cut DNA 2. Estimate the size of the DNA bands that result from restriction digestion using gel
electrophoresis.
3. Identify the various conformations of uncut plasmid DNA as seen in gel electrophoresis.
4. Discuss the different lifestyles of bacteriophage.
5. Estimate the size and concentration of plasmid DNA using gel electrophoresis results.
BACKGROUND Plasmids
Plasmids are circular pieces of DNA that exist independent of the chromosome of prokaryotic cells. Plasmids are retained because they carry genes that benefit the bacterium, such as genes for antibiotic resistance. Plasmid DNA exists naturally as a supercoiled molecule, in which the circle of DNA is folded upon itself, much like a twisted rubber band collapses from a circle to a twisted line (Figure 1a). When plasmids have nicks, or breaks along the sugar phosphate backbone of the DNA, the tension of coiling is relieved and a relaxed circular DNA results. The circle is no longer held together totally by covalent bonding between the sugars and phosphates, but instead is weakly held together by the hydrogen bonding between the base pairs. Plasmids also exist in another type of conformation – that of interlocking rings called catenanes, which form during replication. There can be dimers (two rings), trimers (three rings), and tetramers (four rings). Each of the interlocked circles can be supercoiled (Figure 1a) or relaxed (Figure 1b).
a. b.
Figure 1. Plasmid conformation models; a. super coiled; b. relaxed dimer catenane
When uncut plasmid DNA is run on an agarose gel, many bands are usually visible.
Recall that it is the size of the DNA that determines how quickly it can migrate through a gel in an electric field. The shape of the molecule also has a bearing on the migration – the more compact the shape, the faster it will move. Each of the varying shapes that plasmid DNA can take allows it to move through agarose gel at many speeds. The supercoiled plasmids move fastest and are most abundant, catenanes move more slowly, and open circular or nicked DNA runs somewhere in between. Look for slight bands all along the path that the plasmid DNA travels in the gel. Figure 2 shows three
conformation bands for an uncut 6.2 kb plasmid. Notice that the supercoiled band is most abundant (it is brighter, which indicates more DNA) and it runs at the same speed as a linear fragment of DNA that is just under 4 kb in size. Along with DNA, small fragments of transfer RNA and digested ribosomal and messenger RNA may be present. This is seen as a smear at the bottom of lane 1.
Researchers make use of genetically engineered plasmids as cloning vectors. High copy number plasmids will replicate as many as 500 – 700 times within each cell. Plasmids first engineered at the University of California bear the prefix “pUC” and have been used widely in biotechnology research for the past 30 years.
Cloning Basics
The cloning of a gene first involves restriction enzyme digestion of chromosomal DNA. Restriction enzymes that cut less frequently are chosen, so that the resulting fragments are larger. The more nucleotides in the recognition sequence, the less frequently the sequence will occur, whereas the fewer the number of nucleotides, the more likely the sequence will occur, resulting in an increase in smaller fragments of
Wells
2 kb RNA
Catenanes Nicked circles Supercoiled
Figure 2. Uncut plasmid DNA run on an agarose gel; lane 1 an uncut 6.2 kb plasmid; lane 2 kb Ladder standard DNA marker.
6 kb
1 2
plasmid DNA and at least 1 unit of restriction enzyme/ g of plasmid or phage DNA (about 5-10 units/ g for genomic DNA). Star activity, the indiscriminate cutting of DNA at sites other than the intended recognition sequences, can give abnormal results.
Changes in the buffer cations (Mn
2+, Co
2+, instead of Mg
2+), the cation concentration, the pH of the solution, the presence of glycerol or ethanol in the reaction mixture and excess enzyme can all cause star activity resulting in more DNA fragments than expected. Once the DNA is cut properly, the restriction fragment containing the gene is identified, isolated and purified by a process referred to as Gene Clean . Fragments of the correct size are then joined, or ligated, with larger fragments of DNA called vectors, to create recombinant DNA. The vector DNA can be phage (bacteria-infecting virus), a plasmid, or a combination of the two (cosmids and phasmids). Once the fragments are ligated into the vector, bacterial cells are transformed by manipulations designed to take the
recombinant DNA across cell membranes and into the cytoplasm of the cells. The transformation is plated on selective media and colonies that appear to have been transformed with the recombinant vector are cultured in liquid media overnight. During the growth of the bacterial culture, the gene of interest is cloned within the hundreds of plasmids replicated within each cell, giving enough DNA for further genetic procedures.
The next step is restriction mapping to verify that the plasmid DNA truly contains the gene of interest.
LABORATORY OVERVIEW
In this lab a commercial plasmid will be digested with a restriction enzyme to serve as the vector for recombinant plasmid construction. In successive labs, a fragment of phage DNA will be isolated, purified and then joined to the commercial plasmid, forming a new recombinant plasmid.
TIMELINE:
Day 1 – Set up digestions and run E-gels to confirm digestion; cast 0.8 % gel for the next lab, if time permits.
SAFETY GUIDELINES
Ethidium bromide is a strong mutagen. Gloves must always be worn when handling gels or buffers containing this chemical.
Boiling agarose can cause burns. Wear hot gloves when removing agarose from hot plate or microwave oven.
The electric current in a gel electrophoresis chamber is extremely dangerous. Never remove a lid or touch the buffer once the power is turned on. Make sure the counter where the gel is being run is dry.
UV light, used to illuminate the DNA stained with ethidium bromide, is dangerous. Eye
protection must be used.
MATERIALS - DNA Digestion
pUC 18 DNA, approximately [0.5 μg/μL]; dilute per instructor‟s directions Eco RI endonuclease
10x buffer for Eco RI, 50 µL/team Reaction grade dH
2O
Water bath set at 37 C
automatic micropipetters and tips used tip container
gloves Kim wipes
1.5 ml microcentrifuge tubes microcentrifuge tube rack Sharpie marking pen
Floating microfuge tube racks for water bath MATERIALS - Gel Electrophoresis E-gels and rigs
Uncut pUC 18 DNA as control on gel
Molecular Weight Marker DNA (with 10 L concentration labeled) Sharpie marking pen
gloves dH
2O
automatic micropipetters and tips Used tip container
Kim wipes
1.5 ml microcentrifuge tubes Microfuge tube rack
UV Transilluminator UV Camera and film
UV eye protection and face shields
PROCEDURE
Each team will set up one digestion.
Set the water bath at 37 C.
PART I. DNA Digestion
NOTE: We will be using commercial Lambda DNA cut with Eco RI, so only the pUC 18 plasmid needs to be digested in this lab.
1. Completely thaw the restriction enzyme buffer. DO NOT USE BUFFERS THAT CONTAIN ICE; THE BUFFER CONCENTRATION WILL BE INCORRECT.
2. Add the following to a labeled 1.5 ml microcentrifuge tube to give a total volume of 30 l. Use a new tip for each reagent. (NOTE: A total volume of 30 L is used here so that part can be run on a gel now and the rest can be saved for the recombinant plasmid construction.)
pUC 18 DNA digest:
Molecular grade dH
2O ______ L pUC 18 DNA ( 1.0 g) ______ L 10x buffer [1.0x final] 3.0 L
*Eco RI (1U/ g of plasmid) 0.5 L
Total volume: 30.0 l
* Restriction enzymes are dissolved in glycerol, which is viscous. Take extra care to just touch the pipette tip to the surface, so none of the costly enzyme is wasted on the outside of the pipette tip.
Let the micropipetter plunger up very slowly and hold the tip still to allow the glycerol to enter completely. Wipe the tip on the inside of the tube to save as much as possible, as most enzymes are very expensive. If glycerol appears on the outside of the tip, use a Kim wipe to remove it to prevent Star activity.
3. Spin briefly to mix. Incubate at 37 C in a water bath for at least 1 hour to overnight.
(NOTE: While incubating, pour the gel you will use for the next lab when you perform the gene clean.)
4. Make a table of the expected results from the digestion of pUC 18 and Lambda DNA + Eco RI (even though you will not cut Lambda in this lab). Include this table in the Analysis section of this lab in your lab notebook. (See the sample data table and the sample gel, below, and the maps and information at the end of this lab.)
Table 1. Expected EcoR I Restriction Results
DNA Size expected - uncut Sizes expected – cut with EcoR I pUC 18 plasmid
Lambda DNA genome
Figure 3. Sample Gel of Expected Results
PART II. Casting a 0.8% Gel for DNA Electrophoresis and Gene Clean (next lab) 1. Weigh out 0.4 grams of agarose and place in a 250 ml flask. Add 50 ml 1X TAE
buffer and microwave until completely melted, 1 - 2 minutes.
2. Set up the electrophoresis chambers as in previous lab.
3. When you can hold your hand on the bottom of the flask of heated agarose for 30 seconds, it is cool enough to pour. Check the volume after microwaving and add more dH
2O to return the volume to 50 ml, if necessary. Pour the melted agarose into the casting tray. Insert the combs at the end that will have the negative electrode attached. (To make this determination, fit the lid on the gel box and connect the electrodes to the power supply.)
4. Allow the gel to cool and solidify (about 15-20 minutes). Do not disturb the gel during this time.
5. When the agarose has solidified, gently remove the comb, pulling it straight upwards, in one motion. Wrap the gel in plastic wrap and/or place it in a zip lock bag that you have labeled with your group number and date. Store the gel at 4°C until the next lab.
Lane 1. Mol. Wt. Marker Lane 2. Uncut Lambda Lane 3. Lambda + EcoR I Lane 4. Uncut pUC 18 Lane 5. pUC 18 + EcoR I
Part III. Preparation of pUC 18 digestions, gel loading and running
1. Add 15 µL of molecular grade water to a labeled 1.5 µL microcentrifuge tube. Pulse spin the digested pUC 18 DNA in a microcentrifuge to pull any condensation to the bottom of the tube. Transfer 5 L of this digestion into the labeled microfuge tube.
Place the remainder of the digestion in the class freezer box.
2. Add 10 L of molecular grade water to a fresh tube labeled as „Marker DNA.‟
Transfer 10 µL of the molecular weight marker DNA to this tube.
(The first group to this point must perform the next step – dilution of the control uncut pUC 18. Dilute so that there is approximately 50 ng/20 uL for each E-gel to be run.)
3. Heat all pUC 18 DNA restriction digestions at 65°C for 2 minutes. (NOTE:
Determine if the molecular weight marker being used also must be heated prior to loading; if so, heat along with the DNA restrictions.) Pulse spin and place on ice.
Make a note of the marker DNA you are using. Include a photocopy of information on this marker in your notebook.
4. Insert the E-gel into the holder and run for 2 minutes with the comb still in the wells to activate the ethidium bromide. The prerun is started by pressing down on either start button and holding until there is a beep. The gel rig will signal at the end of the 2 minute prerun. Remove the comb.
5. Do not leave any wells empty – load each with a DNA sample or water. Load the DNA onto the E-gel(s) in the order decided on for the class.
6. Run the gel for 30 minutes, view on the transilluminator, and document for your lab notebook.
7. Before leaving the lab, make sure labels on all tubes are complete. The remainder of each digestion will be placed in the class freezer box and stored at -20 C to be used in the following labs.
DATA ANALYSIS
Determine if the pUC 18 DNA is cut completely. If not, further incubation should yield
complete cutting. The size of the DNA fragments can be estimated by comparing the
distance each fragment has migrated relative to the molecular weight marker bands. Size
determination has a 10% error, so remember that this is an estimate. (Do not graph the
results – just estimate by observing the molecular weight marker and the uncut pUC 18
bands on the gel photo.)
BIOTECHNOLOGY I – DNA RESTRICTION for CLONING
QUESTIONS
1. What is the function of a plasmid and where are they found naturally?
2. Where do DNA restriction enzymes exist naturally and what is their function?
3. Explain why you would cut with a 6-base cutter rather than a 4-base cutter if you were planning to clone a gene from chromosomal DNA.
4. What is Star Activity?
5. What are the three conformations of uncut plasmid DNA that might be seen on a gel?
6. Was the DNA in your digestion completely cut? How did you know whether it was or was not cut?
DNA MAPS
Size = 48,502 bp
Sources:
http://www.accessexcellence.org/AE/AEPC/Wards/restrict/restriction_mapping.html
Lambda DNA cut with EcoR I gel electrophoresis results:
