Biotechniques (Biol 410) 13. DNA Extraction & Gel Electrophoresis

Transcription

1 Biotechniques (Biol 410) 13. DNA Extraction & Gel Electrophoresis Laboratory Objectives Extract DNA from cells using an alternative to Column purification Learn to prepare and pour gel DNA Electrophoresis Gels o Set up electrophoresis apparatus Conduct a restriction digest Separate DNA fragments using Electrophoresis o Analyze results INTRODUCTION Observation of DNA is an important part of molecular and genetics research. Some of the important techniques within molecular genetics include transgenics, genetic manipulation, knockout mutation. Before we move into the realm of molecular genetics we need to become proficient in isolating the DNA for further observation. Once DNA is extracted from the cell we can being to analyze and modify it contents. In our last lab we isolated DNA plasmids from bacteria using column chromatography. Now we will be isolating DNA by an alternative method, alcohol condensation, which takes advantage of Structure of DNA the fact that DNA is insoluble in alcohol. Once DNA is made insoluble, we can condense it for collection and further activities, such as gel electrophoresis. DNA gel electrophoresis uses gel of a set molecular density separate the DNA pieces by size. Restriction digests can be used to cut DNA in specific locations, allowing us to then separate the resulting pieces. In the following two labs we will become proficient in collect DNA, using restriction enzymes, and observing the fragments we extract through gel electrophoresis. DNA Extraction The first step in any genetic analysis requires collecting DNA, which means you need to break open the cell and nuclear membranes in an effective way, without damaging the DNA. This process is called lysing. Most extraction techniques use detergents as lysing agents, which disassociate lipids in the membrane. Sodium Dodecly Sulfate (SDS) and Ethylenediaminetetraacetic disodium salt (EDTA) are two of the most common organic lysis solutions. Mechanical lysing can also be done using silica beads. DNA Extraction by Alcohol precipitation Once membranes have been lysed, proteins must be degraded, in particular histones. Histones are the proteins that support DNA during supercoiling (chromosome formation). By degrading histones, DNA is fully released and can be examine and manipulated. We typically use enzymes to degrade proteins called protease. SDS, proteinase k, Page 1 of 13

2 pronase, and phenol-chloroform are used during protein removal. The process of protein removal is accelerated by exposure to heat. Heat causes proteins to denature, and loose their function, making them easier to degrade. However, if samples are placed in to high a heat, it will cause other molecules to degrade, including DNA. Once the membranes and proteins are degraded the DNA can be extracted. DNA is non-soluble in alcohol, so the addition of alcohol to a sample containing release DNA will cause the DNA to precipitate, making it visible, and easier to collect. Restriction Enzymes DNA splicing, the cutting and linking of DNA molecules, is one of the basic tools of modern biotechnology. The basic concept behind DNA splicing is to remove a functional DNA fragment, let s say a gene, from one organism and to combine it with the DNA of another organism in order to study how the gene works. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes have been given to people with nonfunctional genes, such as those who have a genetic disease like cystic fibrosis. The ability to cut and paste, or cleave and ligate, a functional piece of DNA predictably and precisely is what enables biotechnologists to recombine DNA molecules. This is termed recombinant DNA technology. The first step in DNA splicing is to locate a specific gene of interest on a chromosome. A restriction enzyme is then used to cut out the targeted gene from the rest of the chromosome. This same enzyme is also used to cut the DNA of the recipient into which the fragment will be inserted. Restriction digestion cuts DNA leaving stick ends, which can be used for insertion of new genes (see below). Restriction enzymes also create multiple fragments of DNA that can be separated, quantified, and observed in gel electrophoresis. If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments of DNA. Therefore, if a given piece of linear DNA is cut with a restriction enzyme whose specific recognition sequence is found at five different locations on the DNA molecule, the result will be six fragments of different lengths. The length of each fragment will depend upon the location of restriction sites on the DNA molecule. Restriction enzymes are proteins that cut DNA at specific sites. Restriction enzymes, also known as restriction endonucleases, recognize specific sequences of DNA base pairs and cut, or chemically separate, DNA at that specific arrangement of base pairs. They were first identified in and isolated from bacteria that use them as a natural defense mechanism to cut up the invading Page 2 of 13

3 DNA of bacteriophages viruses that infect bacteria. Any foreign DNA encountering a restriction enzyme will be digested, or cut into many fragments, and rendered ineffective. These enzymes in bacteria make up the first biological immune system. There are thousands of restriction enzymes, and each is named after the bacterium from which it is isolated. For example: EcoRI = The first restriction enzyme isolated from Escherichia coli bacteria HindIII = The third restriction enzyme isolated from Haemophilus influenzae bacteria PstI = The first restriction enzyme isolated from Providencia stuartii bacteria Each restriction enzyme recognizes a specific nucleotide sequence in the DNA, called a restriction site, and cuts the DNA molecule at only that specific sequence. Many restriction enzymes leave a short length of unpaired bases, called a sticky end, at the DNA site where they cut, whereas other restriction enzymes make a cut across both strands creating double-stranded DNA fragments with blunt ends. In general, restriction sites are palindromic, meaning the sequence of bases reads the same forwards as it does backwards on the opposite DNA strand. Lambda Phage DNA Lambda DNA is the genomic DNA of a bacterial virus, or bacteriophage (phage), which attacks bacteria by inserting its nucleic acid into the host bacterial cell. Lambda is a phage that replicates rapidly inside host cells until the cells burst and release more phages to carry out the same infection process in other bacterial host cells. Bacteriophage lambda is harmless to man and other eukaryotic organisms, and therefore makes an excellent source of DNA for experimental study. Lambda DNA has also been well studied so we can use it for comparison to our ampicillin plasmid DNA that we collected previously. he sites where e, here is a G A-A-T-T-C EcoRI C-T-T-A-A G HindIII Electrophoresis & making DNA visible A DNA fragment that has been cut with restriction enzymes can be separated using a process known as agarose gel electrophoresis. The term electrophoresis means to carry with electricity. Agarose gel electrophoresis separates DNA fragments by size. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole (anode) when placed in an electric field. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs. Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single bands of DNA. These bands will be seen in the gel after the DNA is stained. DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. A loading dye containing two blue dyes is added to the DNA solution. The loading dye does not PstI e DNA A A-G-C-T-T T-T-C-G-A A C-T-G-C-A G G A-C-G-T-C Diagram of restriction sites Diagram of a Lambda phage virus Page 3 of 13

4 stain the DNA itself but makes it easier to load the gels and monitor the progress of the DNA electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. The faster dye co-migrates with DNA fragments of approximately 500 bp, while the slower dye co-migrates with DNA fragments approximately 5 kb in size. Staining the DNA pinpoints its location on the gel. When the gel is immersed in Fast Blast DNA stain, the stain molecules attach to the DNA trapped in the agarose gel. When the bands are visible, your students can compare the DNA restriction patterns of the different samples of DNA. METHODS Lab 1 DNA electrophoresis gel demonstrating restriction digestion to create DNA fragments Pouring 1% Agarose Gel 1. Mix 1.5g of agarose powder into 150ml TAE buffer to create a 1% agarose solution (this may already be done for you). Agarose will quickly congeal. 2. Microwave for 3 min, stop at 90 seconds to swirl and make sure agarose is suspended (CAUTION: Hot! Use gloves). 3. Be careful to watch agarose to stop microwave when agarose first begins to boil. 4. Let sit for 1-5 minutes (until almost bearable to touch with bare hands). 5. Then pour gel into the gel mold with 2 8-lane combs. a lane combs for lab 2 6. Let cool for minutes, agarose will become cloudy. Then it can be removed from the molding chamber 7. See Tips and Tricks for additional methods of preparing agarose for DNA electrophoresis Prep of column chromatography samples (last lab) 1. Gather 10µl of your column chromatography sample into a new 1.5ml microfuge tube. 2. Add 2µl loading dye. 3. Flick or vortex to mix and tap on desk (or pulse centrifuge) to collect at the bottom of the tube. Page 4 of 13

5 DNA Extraction Modified from: T. Maniatis, E.F. Fritsch, J. Sambrook (1982) Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory, p Inoculate 6ml of medium containing the appropriate antibiotic (10µl amp) with a single bacterial colony. Incubate at 37 o C overnight with vigorous agitation. a. This has been done for you. 2. Pour 1.5ml of the culture into an Eppendorf tube. Centrifuge for 1 min at 6000RPM. 3. Remove supernatant solution with a pipet. 4. Repeat steps 2 & 3 until all 6ml of the bacterial culture has been centrifuged. 5. Re-suspend the cell pellet in 0.35ml of STET solution 6. Add 25µl of freshly prepared solution of lysozyme (10mg/ml in 10mM Tris HCl, ph 8.0). Mix by vortexing for 3 sec. Lysozyme is an enzyme, which breaks down the carbohydrates in bacterial cell walls. 7. Incubate on ice for 10 min. 8. Place tube in boiling water 1-2 min. 9. Microfuge for min at 6,000rpm. 10. Transfer supernatant solution to a new tube 11. Treat pellet with 20µl RNase for 10 min at 37 o C 12. Add 40µl of 2.5M sodium acetate and 420µl of isopropanol. 13. Incubate at 20 o C for min 14. Microfuge for 5 min at 6000rpm 15. Decant off supernatant 16. Wash with 500µl of 75% EtOH a. This replaces isopropanol, evaporates better and washes away salt. b. Sample can be stored in fridge for several days 17. Remove EtOH and invert tube on kimwipe 18. Re-suspend in 50µl in sterile water 19. Transfer 10µl to a new 1.5ml microfuge tube a. Place the remainder of your sample on ice, and save for next lab. Page 5 of 13

6 20. Add 2µl loading dye to your sample, and mix vigorously or vortex. Tap your sample or briefly vortex to collect the sample at the bottom of the tube. 21. Proceed to setting up your gel. Setting up Gel Electrophoresis 1. Obtain a micro test tube of DNA marker (M). 2. Place agarose gel in the electrophoresis chamber add 1x TAE buffer so it fills the wells at both ends, and just covers the gel. a. Make sure the wells are located near the black ( ) electrode and the bottom edge of the gels is near the red (+) electrode. Remember DNA moves from to Gently remove the combs gently so as to not tear the gel (ask the Professor for help if necessary). 4. Load 5µl of molecular ladder (Standard Marker) in the first lane. 5. Load 10µl of each sample into separate wells in the gel chamber. a. Be sure to keep track of the lane order in which you load your samples. 6. (Lab 1 only) We are running samples from the column chromatography lab and this weeks DNA extraction. a. This DNA is a circular plasmid and has not been digested So this is a test to see if we collected DNA. b. Load chromatography samples on the top row (wells nearest the black terminal) and This weeks DNA extraction samples on the bottom row (wells closest to the red terminal end). Make sure to keep track of which sample is yours 7. (Lab 2) Both groups will share a gel. Group 1 will load their samples on the top row (closest to the black terminal), and Group 2 will load their samples on the bottom row (closest to the red terminal) 8. Carefully place the lid on the electrophoresis chamber. Connect the electrical leads into the power supply, red to red and black to black. 9. Turn on the power and run the gel at 100 V for minutes. a. Gels should run until the sample is at least 3/4 the distance to the next set of lanes, or the bottom of the gel. Orientation of the agarose gel relative to the gel box electrodes. DNA has a slight negative charge, and will be repelled away from the negative pole, and attracted towards the positive, causing the DNA to migrate Page 6 of 13

7 10. When the electrophoresis run is complete, turn off the power and remove the top of the chamber. Carefully remove the gel and tray from the gel box. Be careful the gel is very slippery. Slide the gel into the staining tray. 11. Add 120 ml of 100x Fast Blast stain into a staining tray. Place on the shaker a. Alternatively samples can be stained in 1x Fast Blast stain over night. No destaining required. 12. Stain the gels for 2 minutes. 13. Rinse the gel with warm tap water until water runs clear. Fill the tray and return to the shaker. 14. De-stain for 5 minutes each with gentle shaking for best results. 15. Rinse with warm tap water, and de-stain on the shaker for another 2 minutes. 16. Transfer the gel to the light box and record the results. a. Photograph, draw a diagram, or both b. Photograph using the gel documenter in the research space 17. Using a ruler measure the distance each band traveled from the well. Using the Marker lane to estimate the fragment size in each lane. Page 7 of 13

8 Lab 2 Restriction Enzyme Digest 1. Obtain micro test tubes that contain each (1 per pair): a. EcoRI enzyme stock solution (green) b. HindIII enzyme stock solution (orange) c. PtlI enzyme stock solution (violet) d. Lambda DNA (yellow) e. Your Plasmid DNA from last two labs f. Restriction buffer (RB) g. Keep all solutions on ice. 2. Using a fresh tip for each sample, pipet the reagents into 8 clean micro test tubes according to the table below: Tube DNA Buffer PstI EcoRI HindIII 1 Lambda - 4µl 6µl Lambda - 4µl 5µl 1µl Lambda - 4µl 5µl - 1µl - 4 Lambda - 4µl 5µl - - 1µl 5 Column Plasmid - 4µl 6µl Column Plasmid - 4µl 5µl 1µl Column Plasmid - 4µl 5µl - 1µl - 8 Column Plasmid - 4µl 5µl - - 1µl 9 Extraction Plasmid - 4µl 6µl Extraction Plasmid - 4µl 5µl 1µl Extraction Plasmid - 4µl 5µl - 1µl - 12 Extraction Plasmid - 4µl 5µl - - 1µl 3. Mix the components by gently flicking the tube with your finger and tapping gently on the table to collect liquid to the tube bottom. Collect all the liquid to the bottom of the micro test tube by tapping them gently on the bench top. 4. Place the tubes in a floating rack and incubate for 30 minutes at 37 C. a. While tubes are incubating pour your gel (see below) 5. After the incubation, place the samples back on ice. Or Samples can be stored at 4 o c. 6. Since we are going to run our samples today, add 2µl of sample loading dye into each tube. 7. Mix the contents by flicking the tube with your finger. Collect the sample at the bottom of the tube by tapping it gently on the table. 8. Proceed to setting up gel and loading samples (same general procedure as last week). a. Load Molecular marker in 1 st lane. Page 8 of 13

9 Measuring DNA fragment lengths One of the first steps to analyze your data is to determine the approximate size of each of your restriction fragments. This can be done by comparing the DNA restriction fragments with DNA fragments of known sizes, called standards. 1. Using a ruler, measure the distance (in mm) that each of your DNA fragments (bands) traveled from the well. Measure the distance from the bottom of the well to the bottom of each DNA bands, and record the numbers on the table. Repeat for each of your restriction cut samples. 2. To estimate size in base pairs (bp) of each restriction fragment measure the distance traveled of each band in the DNA marker/standard. 3. Graph the distance traveled (x-axis) and size of the band in BP (y-axis) to create a standard curve a. Graph by hand on logarithmic graph paper (last page) or using excel. b. Add a linear trend line x = Known fragment size o = Unknown fragment size 8,000 Size, base pairs 7,000 6,000 5,000 4,000 3,000 Unknown fragment 1 Unknown fragment 2 2,000 1,000 Distance traveled, mm Set the y-axis to a logarithmic scale, as distance travel is exponential. Smaller bands move much faster (order of magnitude) than larger bands. Page 9 of 13

10 4. Compare the distances that the bands of known sizes traveled, relative to your samples. a. Record the results for Lambda DNA below, or in your lab notebook (From the top) DNA Marker Distance in mm Actual BP Undigested LDNA Distance in mm Actual BP PstI digested LDNA Distance in mm Actual BP EcoRI digested LDNA Distance in mm Actual BP HindIII digested DNA Distance in mm Actual BP Band 1 23,130 Band 2 9,416 Band 3 6,557 Band 4 4,361 Band 5 2,322 Band 6 2, Repeat for Column and Extraction Bacteria Plasmids, and record in your lab notebook. a. Include these tables in your report Follow up Questions How can restriction enzymes be used to identify DNA fragments? How many fragments could you cut your Ampicillin resistant plasmid into with each restriction enzyme? o What does this tell you about your plasmid? Are the two plasmids we extracted the same? o How can you tell? Page 10 of 13

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12 Additional Tips and Tricks for Making Agrose Gels 1% agarose - Gel Prep 1 gram of agarose for each 100 ml of 1x TAE electrophoresis buffer for 1% gel o Higher concentration gels (up to 5%) are used to separate DNA very close in size, but take a long time to separate DNA. Add the agarose powder to a suitable container, preferable an Erlenmeyer Flask (You usually want the final agarose solution to fill ¼ to ½ of the container). Add the appropriate amount of 1x TAE electrophoresis buffer and swirl to suspend the agarose powder in the buffer. o If using an Erlenmeyer flask, invert a second 25 ml Erlenmeyer flask into the open end of the Erlenmeyer flask containing the agarose. The small flask acts as a reflux chamber, thus allowing long or vigorous boiling without much evaporation. The agarose can be melted for gel casting by boiling until agarose has melted completely on a magnetic hot plate, hot water bath, or in a microwave oven. o DO NOT OVERBOIL!!! Microwave Boiling Microwave for 3 minutes, stopping every minute to swirl flask and make sure agrose is suspended (CAUTION: Hot! Use gloves). Be careful to watch agarose to stop microwave when agarose first begins to boil. Let sit for 1-5 minutes (until almost bearable to touch with bare hands. Then pour gel. Hotplate Boiling Add a stir bar to the un-dissolved agarose solution. Heat the solution to boiling while stirring on a magnetic hot plate. o Bubbles or foam should be disrupted before rising to the neck of the flask. Boil the solution until all of the small transparent agarose particles are dissolved. Set aside to cool to C before pouring gels. Page 12 of 13

13 100,000 80,000 Semilog Graph Paper 60,000 40,000 20,000 10,000 8,000 6,000 Size, base pairs Size, base pairs 4,000 2,000 1, Distance traveled, mm Page 13 of 13