BIO440 Genetics Lab Humboldt State University Gel Electrophoresis: Quantitative length and mass measurements of DNA Electrophoresis, and in particular agarose gel electrophoresis, is an integral analysis used in all aspects of genetic studies. This analysis allows you to determine the size of DNA fragments. It also allows you to determine the mass of DNA fragments. This experiment is designed to expose you to a variety of skills and methods involving agarose gel electrophoresis. Objectives: To give you experience setting up, running, and analyzing agarose gels. To allow you to determine the success of your PCR amplification, PCR product purification, and PCR product digestion with restriction enzymes. To allow you to identify unique 16S rrna gene clones for DNA sequencing. Approach: Each group will run 10 samples on a 1.5% agarose gel: 1-8 10 µl of each of your individual PCR products. Lanes 1-6 are derived from white colonies, lane 7 from a blue colony, and lane 8 is your no-template-dna negative control 9- a molecular weight marker, 100 bp ladder these samples will be used for analyzing the length of PCR products 10- bacteriophage lambda DNA, pre-digested with HinDIII restriction enzyme these samples serve as molecular weight and mass standards In addition, a separate 3% agarose gel will be run to compare the fragments resulting from restriction digestion of each of the 48 PCR products. Introduction: Agarose Gel Electrophoresis Electrophoresis is used to separate different types of molecules from one another. The common theme is that you set up a matrix such that diffferent types of molecules move through the matrix at different rates. In the case of agarose gel electrophoresis of DNA we can separate molecules based on their size (also referred to as their length, or the number of base-pairs). DNA has a negative charge in solution (remember the phosphodiester backbone), so it will migrate to the positive pole in an electric field. In agarose gel electrophoresis the DNA is forced to move through a sieve that is made of agarose (a purified sugar extracted from seaweed). Large pieces of DNA have a more difficult time achieving net movement through the tortuous path of an agarose sieve, and therefore move more slowly than small pieces of DNA. The actual size of a DNA molecule can be inferred by comparing the distance that an unknown DNA molecule moved with the distance that known standards moved. In order to visualize the DNA, it is stained with ethidium bromide and exposed to UV light. The ethidium bromide that has intercalated into the DNA molecule will absorb the UV light and fluoresce. Different concentrations of DNA are useful for isolating different-sized DNA fragments. The table below depicts this relationship.
% agarose Size of fragments separated (kbp) 0.3 60-5 0.6 20-1 0.7 10-0.8 0.9 7-0.5 1.2 6-0.4 1.5 4-0.2 2.0 3-0.1 We can also quantify the mass of our DNA samples. We earlier quantified DNA in solution by measuring UV absorbance at 260 nm on a spectrophotometer capable of light emission in the UV range. The samples were diluted, placed in a quartz cuvette and the A260 and A280 readings taken. Absorbance at 280 nm was done to detect proteins and other contaminants. Proteins absorb strongly at 280 nm due to the presence (mainly) of tryptophan. Typically, pure nucleic acids will have a 260:280 ratio of ~ 1.8. If the ratio falls below this range, the DNA solution contains impurities. A solution of 50 µg DNA/ ml will have an OD 260 of 1.0. Thus, the concentration of DNA in an unknown sample can be determined by the following equation: Ci = (OD260 x 50 µg/ml) / dilution. However, good spectrophotometers are not always available, many cuvettes require large sample volumes, and most importantly, spectrophotometers don't differentiate between degraded and undegraded DNA. Therefore it is often preferable to estimate DNA concentrations by gel electrophoresis and comparison of band intensities with a standard DNA of known concentration. Of course, this method has its own limitations when applied to some samples. For this we will use a phage called Lambda (λ) which has been cut by the restriction enzyme HindIII. We will use HindIII-cut λ DNA as a source of molecular weight markers for our gels. This digest produces 8 bands, having sizes of 125, 564, 2027, 2322, 4361, 6557, 9416, and 23130 base pairs (bp). The smallest fragment will probably not be visible on your gel, however --it may run off the bottom of the gel and even if it doesn t, it probably won t have enough mass to be visible (minimum mass visible on ethidium bromide gel is about 5 ng). You will need to heat this molecular weight marker at 65 C for 10 min before using it, since lambda can exist in both circular and linear forms and hence two of the fragments contain complementary sites which can undergo base-pairing. Introduction: Restriction digests In 1978, Daniel Nathans, Hamilton Smith, and Werner Albert received the Nobel Prize in Physiology and Medicine for their work on enzymes that cut DNA at specific sequences of nucleotides. These enzymes, known as restriction endonucleases, are critical to any studies involving gene cloning and analyses as they allow you to cut up larger pieces of DNA into smaller fragments with known ends. A subgroup of these endonucleases (type II endonucleases) recognize short nucleotide sequences and make double stranded cuts within (or adjacent to) their recognition sequence. Often, recognition sequences are characterized by a region of dyad symmetry (i.e. the 5 to 3 nucleotide sequence of one DNA strand is identical to that of the complementary strand). Several (>600) type II restriction endonucleases have been isolated 2
from prokaryotes. They are presumed to function in degrading foreign DNA, for example as might be encountered from an invading bacteriophage. Bacteria protect their own DNA from being degraded by covalently attaching a methyl group to adenines or cytosines within the recognition sequence. An example of a restriction enzyme is EcoRI. This restriction enzyme was the first restriction enzyme isolated from Escherichia coli strain R, and the name of the enzyme reflects this information. Similarly, the restriction enzyme HinDIII was the third restriction enzyme isolated from Haemophilus influenza strain D, and SalI was the first restriction enzyme isolated from Streptomyces albus. Each of the above restriction enzymes cuts both strands of double-stranded DNA molecules at the enzymes' recognition sequences. Enzyme Name Organism Isolated from Recognition Sequence/ Position of Cut EcoRI Escherichia coli strain R 5' G^ A A T T C 3' 3' C T T A A^G 5' HinDIII Haemophilus influenza strain D 5' A^ A G C T T 3' 3' T T C G A^A 5' SalI Streptomyces albus 5' G^ T C G A C 3' 3' C A G C T^G 5' PstI Providencia stuartii 5' C T G C A^G 3' 3' G^A C G T C 5' SmaI Serratia marcesens 5' CCC^ GGG 3' 3' GGG^ CCC 5' With the exception of SmaI, all of the above enzymes cut the different strands of DNA such that small regions of single-stranded DNA result. For example, if the below fragment were digested with EcoRI... 5' A A A A A A A T G C G A A T T C A A A A A A A A A A A C T G G 3' 3' T T T T T T T A C G C T T A A G T T T T T T T T T T T G A C C 5' 5' A A A A A A A T G C G 3' 5' A A T T C A A A A A A A A A A C T G G 3' 3' T T T T T T T A C G C T T A A 5' 3' G T T T T T T T T T T G A C C 5' These single-stranded regions are sometimes called sticky ends, and are useful in constructing recombinant plasmids in cloning experiments. For example, as mentioned above, plux was 3
constructed by digesting V. fischeri genomic DNA and pgem plasmid DNA with the enzyme Sal I, and then re-ligating the pieces such that a large Sal I fragment of V. fischeri genomic DNA was cloned into the Sal I site of pgem. Protocols: agarose gel electrophoresis Preparation of gel 1. Your instructor will demonstrate how to prepare a gel mold. The wells of the gel are formed by a comb. Each tooth on the comb creates a well (or lane) for each individual sample; our combs create lanes for 10 samples or for 14 samples. 2. We are going to make 60 ml volume, 1.5% agarose gels. In a 250 ml flask, measure out the proper amount of agarose. How much will you use? After adding the buffer, the agarose is heated in the microwave until all of the agarose is in solution. The flask is then allowed to cool several minutes before pouring the molten solution into the mold. Pouring the very hot agarose into the plastic gelbox can cause the (expensive) gel boxes to crack. After the gels are poured - let them solidify for ~ 15 minutes. 3. Once the gel has solidified, the comb will be carefully removed from the gel, and enough buffer will be added to just cover the gel. Make sure that all wells are completely filled with buffer, and that the buffer just barely covers the gels. Running and analyzing gel We will run the gels at a constant voltage of 90-100 V (~6 V / cm gel). After the gel has run about 1 hour, we will stain the gel in ethidium bromide and visualize the bands on a UV transilluminator. We will then take a picture of the gel. *****(Ethidium Bromide =TOXIC, NASTY, MUTAGENIC...OBSERVE STRICT SAFETY PRECAUTIONS!!!!!!!!!) The first step is to load the samples in the wells. Prior to loading, each sample is mixed with loading dye. The loading has glycerol in it which keeps the sample from diffusing out of the well. The loading dye also has separate dyes in it that allow you to estimate how far your sample has run. Each sample should be prepared according to the formula below before loading onto the gel. 3 µl 10X loading dye + 10 µl (this can vary) sample DNA + µl water = 18µl The one exception to this will be the PCR product digests that you prepared. You carried out these digests in a volume of 25 µl, and you want to run that entire volume. Before loading your sample, add 3µl of loading dye to the digest in the tube. You will load the entire volume (25 µl + 3 µl loading dye) of these samples. You will also run a sample of predigested lambda DNA (cut w/ Hind III restriction enzyme) as a size standard. Two different masses of this marker are usually run, and will 4
be if there are enough wells available. To prepare the standard, HinDIII-cut lambda DNA was combined with TAE buffer and loading dye. Heat this mixture at 65 C in heat block for 3-5 minutes, then immediately put on ice until loading. Although isolated lambda DNA is generally linear, it is circular inside a cell and has sites which promote spontaneous annealing at cool temperatures. Heating at 65 C and "snap cooling" on ice will prevent restricted fragments from rejoining. Failure to heat/cool may cause the 23 kb and 4.4 kb fragments to run as a single band (above the 23kb fragment). If you wish, we can run an unheated sample of marker to demonstrate this phenomenon. The table below depicts how you should load your samples if you prepare the gel using a 14-tooth comb. If you used a comb that only has ten wells, then eliminate the 500 ng marker. Lane # Description of Sample Loaded 1 10 µl Lowest #'d purified white PCR product 2 10 µl next lowest #'d purified white PCR product 3 10 µl next lowest #'d purified white PCR product 4 10 µl next lowest #'d purified white PCR product 5 10 µl next lowest #'d purified white PCR product 6 10 µl highest #'d purified white PCR product 7 10 µl blue colony PCR product (unpurified) 8 10 µl negative control PCR product (unpurified) 9 10 µl 100 bp ladder 10 100 ng λ HinD III molecular weight markers 11 500 ng λ HinD III molecular weight markers Some thoughts if you do not get the expected results... 1. It isn't very useful to say "It didn't work", and stop there. It will be much more helpful to try to figure out specifically what worked, specifically what didn't, and what some potential causes are -- this will help you to figure out what to change when you do it next time. I.e., - was the plasmid there, but then it disappeared in your restriction digests? 2. It isn't very useful to say " I did everything exactly like I was supposed to!". This almost certainly isn't true of someone starting out, and it does not lead to the identification and correction of whatever did go wrong. 5
Some pointers... In class we talked about the order of addition of reagents, and about keeping things on ice, and about moving fast. We said that it was important to add the reagents as follows: water first - this gives you a large volume to add your tiny droplets of enzymes and DNA to. Buffer second - this keeps the DNA from autohydrolyzing, and provides BSA (Bovine Serum Albumen) to serve as decoy protein. The BSA will bind to any nonspecific binding sites on the tubes that would otherwise sequester your enzyme. Also, if proteases are present the BSA may keep them busy long enough to prevent your enzymes from being degraded. when adding additional reagents, mix gently by pipetting up and down. In general, you need to be aware that DNAses (enzymes that degrade DNA) are all over your hands, the bench, --anything that is not sterile. Don't let your hands touch anything that will come into contact with your reaction -- i.e, -- don't handle tubes by the inside of the cap -- don't tighten a loose pipette tip by holding it in your hand - discard it and put a new one on, tighter. -- don't reach into a tub of tubes to pull out a tube - pour a few tubes into the lid of the container, and then carefully remove the tubes that you need without contaminating the tubes you remove, the other tubes that are present, or the container. 6
Name: Observations and Analyses -Agarose Gel Electrophoresis Tape a copy of the photos of the PCR screen gel below. Label to indicate which lanes contain what plasmid #'s PCR product, and what lanes were used for plasmid preparations for DNA sequencing. Below the gel, briefly interpret the results. 1. On the pgem T-vector with no insert (3018 basepairs) the plasmid is linearized and T- tailed between nucleotide positions number 60 and 61. The PCR screen primer sites we used: "T7" and "M13 reverse" are found at nucleotide positions numbered 2987-3006 and 176-192 respectively. What size PCR product would you expect using these primers to amplify from uninserted circular plasmid? 2. What size PCR product would you expect using these primers to amplify from a colony containing a plasmid inserted with your 16S rrna gene PCR product? 7
3. On the PCR screen gels, estimate the PCR product sizes in your lanes. How do they agree with or differ from your expectations? If they differ, suggest an explanation. 4. Analyze the mass of DNA that is present in each of the PCR product bands, by comparing band intensity of the lambda HinDIII markers with the band intensity in your other lanes Plasmid # Approximate mass of PCR product (ng) Show calculations here: 8
5. Describe the appearance of your purified, digested PCR products on the 3% agarose gel. Are these similar to or different than what you expected? How? 6. Interpret the results of the restriction digest of your PCR products using the table below. Plasmid # Number of bands observed Approximate size of bands (lowest to highest) in bp 7. Based on the gel, which ten plasmids do you think that we ought to prepare for DNA sequencing? 9
8. Troubleshooting. Sometimes, things go wrong. Below is a list of possible (unexpected) ways that your PCR product restriction digestion gel could have looked. Based on the described results, what could have gone wrong in each case? What should you do to repair the damage-- for example, do you need to re-do the analysis? If so, what should you do differently when repeating the analysis? Unexpected Result all of the lanes on the gel, including the markers, had no visible DNA. Your undigested PCR product gel looked fine. What might be wrong? What should you do? the marker lanes had visible DNA, but none of the other lanes had any DNA. Your undigested PCR product gel looked fine. the marker lanes had visible DNA, but none of the other lanes had any DNA. Your undigested PCR product gel also didn't show any DNA bands. the digest lanes looked identical to the uncut lanes Only smears of DNA (no distinct bands) were visible in the PCR product digestion lanes 10