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Isolation and Purification of Total Genomic DNA from E. coli

INTRODUCTION The isolation and purification of DNA from cells is one of the most common procedures in contemporary molecular biology and embodies a transition from cell biology to the molecular biology; from in vivo to in vitro, if you prefer. DNA was first isolated as long ago as 1869 by Friedrich Miescher while he was a postdoctoral student at the University of Tbingen. Miesher obtained his first DNA, which he referred to it as nuclein, from human leukocytes washed from pus-laden bandages amply supplied by surgical clinics in the time before antibiotics. He continued to study DNA as a professor at the University of Basel, but switched from leukocytes to salmon sperm as his starting material. Meishers choice of starting material was based on the knowledge that leukocytes and sperm have large nuclei relative to cell size. DNA isolated from salmon sperm and from (bovine) lymphocytes is still available commercially. Molecular biologists distinguish genomic DNA isolation from plasmid DNA isolation. If you have taken the Biology 20L class you have done plasmid DNA isolation from E. coli using a procedure based on a commercial kit from Promega (Wizard Miniprep System). Plasmid DNA isolation is more demanding than genomic DNA isolation because plasmid DNA must be separated from chromosomal DNA, whereas a genomic DNA isolation needs only to separate total DNA from RNA, protein, lipid, etc. Many different methods are available for isolating genomic DNA, and a number of biotech companies sell reagent kits. Choosing the most appropriate method for a specific application demands consideration of the issues below. No single method addresses all these issues to complete satisfaction. SOURCE: What organism/tissue will the DNA come from? Some organisms present special difficulties for DNA isolation. Plants cells, for example, are considerably more difficult than animal cells, because of their cell walls, and require special attention. Most lab strains of E. coli are fairly straightforward, but a few E. coli strains produce high molecular weight polysaccharides that co-purify with DNA. You need to look into the genotype of the E. coli strain to know whether you need special steps to eliminate this extraneous material. Inasmuch as our E. coli isolates are direct from nature, we are relying on our procedure to deal with this issue. YIELD: How much DNA do you need? If the source is limited, you will need to use a method that is very efficient at producing a high yield. Fortunately, E. coli is easy to grow, and PCR is effective with very small amounts of DNA sample, so this will not be a major issue for us. PURITY: What level of contaminants (protein, RNA, etc.) can be tolerated? The purification method must eliminate any contaminants that would interfere with subsequent steps. This depends, of course, on what you plan to do with the DNA once you have isolated it. PCR will tolerate a reasonable degree of contamination so long as the contaminants do not inhibit the thermostable DNA polymerase or degrade DNA. We also need to strip proteins off the DNA so that it is a good template for replication.

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INTEGRITY: How large are the DNA fragments in our genomic preps? HMW DNA is notoriously fragile. It is easily cut into smaller pieces by hydrodynamic shearing forces and by DNases. Hydrodynamic shear is minimized by avoiding vigorous vortexing and pipetting of DNA solutions. A simple precaution is to use micropipette tips with orifices larger than usual (wide bore tips). The DNases liberated from the lysed cells are usually inactivated by the protein denaturation step in the procedure. Occasionally DNases are introduced to the procedure as accidental contaminants of other reagents, particularly RNase. Many investigators buy special "Molecular Biology" grade reagents that have been certified "DNase-free" by the manufacturer. These are expensive. DNases present as contaminants in RNase solutions can be inactivated by boiling the RNase for 15 minutes. ECONOMY: How much time and expense are involved? For example, CsCI density-gradient ultracentrifugation provides highly pure DNA samples in relatively high yield, and was formerly widely used. However, ultracentrifugation is very expensive because it requires an instrument costing around $ 40,000. Additionally, it is inconvenient because the centrifugation runs typically go many hours. So this method is now used mostly in situations where high yield and high purity are critical. Many biotech companies sell kits with all the reagents necessary for genomic preps. You need to look carefully at the cost of these kits relative to the labor that they save. SAFETY Inasmuch as DNA isolation methods are designed to break cells and denature proteins, it is not surprising that some reasonably nasty reagents are involved. A phenol/chloroform reagent widely used in DNA purification is notoriously hazardous. In fact, phenol/chloroform is probably the most hazardous reagent used regularly in molecular biology labs. Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. So, phenol/chloroform is a double whammy. It is not only dangerous, but expensive, when you consider the cost of hazardous waste disposal. Our procedure does not use phenol/chloroform. LYSIS Cell walls and membranes must be broken to release the DNA and other intracellular components. This is usually accomplished with an appropriate combination of enzymes to digest the cell wall (usually lysozyme) and detergents to disrupt membranes. We use the ionic detergent Sodium Dodecyl Sulfate (SDS) at 80C to lyse E. coli. REMOVAL OF PROTEIN, CARBOHYDRATE, RNA ETC. RNA is usually degraded by the addition of RNase. The resulting oligoribinucleotides are separated from the high molecular weight (HMW) DNA by exploiting their differential solubilities in non-polar solvents (usually alcohol/water). Proteins are subjected to chemical denaturation and/or enzymatic degradadtion. The most common technique of protein removal involves denaturation and extraction into an organic phase consisting of phenol and chloroform. Another widely used purification technique is to band the DNA in a CsCl density gradient using ultracentrifugation.

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Procedure WEAR GOGGLES AND GLOVES DISCARD REAGENTS AND TUBES IN LABELED WASTE CONTAINERS 1. Briefly vortex the overnight culture to ensure that cells are uniformly suspended. Then transfer 1.0 ml of the overnight culture to a 1.5ml microcentrifuge tube.

2. Centrifuge at 15,000 g (or max. speed) for 2 minutes to pellet the cells. Place your tubes opposite each other to balance to rotor. Do not initiate a spin cycle until the rotor is fully loaded; this minimizes the total number of runs required. A cell pellet should be visible at the bottom of the tube. 3. Transfer the supernatant back into the culture tube it came from and discard this culture tube as biohazard waste. Carefully remove as much of the supernatant as you can without disturbing the cell pellet. The pellet may be on the side of the tube, not squarely on the bottom. I use my P1000 set to 950 ul. 4. Resuspend the cell pellet in 600l of Lysis Solution (LS). Gently pipet until the cells are thoroughly resuspended and no cell clumps remain. LS contains the anionic detergent sodium dodecyl sulphate (SDS) to disrupt membranes and denature proteins. You may notice that the cell suspension is not as turbid as the cell culture you started with; this is because some cell lysis has already occurred. 5. Incubate at 80C for 5 minutes to completely lyse the cells. The samples should now look clear. 6. Cool the tube contents to room temperature. Do not rely on temperature equilibration with ambient air. Place the tube in a room temperature water bath for several minutes. 7. Add 3l of RNase solution to the cell lysate. Invert the tube 25 times to mix. 8. Incubate at 37C for 30 minutes to digest RNA. Cool the sample to room temperature. This step is intended to degrade RNA into small fragments or individual ribonucleotides. 9. Add 200 l of Protein Precipitation Solution (PPS) to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds. Do not skimp on the vortexing

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10. Incubate the sample in an ice/water slurry for 5 minutes. The sample now has significant whitish insoluble material. 11. Centrifuge at 15,000 g (or max. speed) for 3 minutes. There should be a large pellet of whitish gunk on the bottom and sides of the tube. The gunk consists of denatured proteins and fragments of membrane and cell wall. 12. Transfer the supernatant (800 l) containing the DNA to a clean 1.5ml microcentrifuge tube containing 600l of room temperature isopropanol (IPA). Be sure that you dont suck up and transfer any of the grungy precipitate. I use my P1000 set to 750 ul. 13. Mix the DNA solution with the IPA by inverting the tube at least 15 times. The DNA is usually (barely) visible as a small floc of whitish material. 14. Centrifuge at 15,000 g (or max. speed) for 2 minutes. 15. Carefully pour off the supernatant (do not pipette) and invert the tube on clean absorbent paper to drain for 2-5 minutes. You want the paper to wick off the IPA that drains down and collects at the rim of the inverted tube. The DNA pellet may or may not be visible. Do not allow the DNA pellet to completely dry. 16. Add 600l of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet. Do not resuspend by pipetting. 17. Centrifuge at 15,000 g (or max. speed) for 2 minutes. 18. Carefully pour off the ethanol supernatant (do not pipette) and invert the tube on clean absorbent paper to drain. You want the paper to wick off the ethanol that drains down and collects at the rim of the inverted tube 19. Allow the pellet to air-dry for 1015 minutes. You want to evaporate as much of the ethanol as possible without letting the DNA pellet completely dry. When all the EtOH is gone there should still be some water left hydrating the DNA. 20. Add 100l of DNA Rehydration Solution (RH) to the tube and rehydrate the DNA by incubating at 65C for 1 hour. After 30 minutes, flick the bottom of the tube gently to facilitate dissolution and mixing. 4 of 6

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Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4C, preferably on a low speed shaker. 21. Analyze the DNA prep. by spectrophotometry using the Nanodrop spectrophotometer and then store the DNA at 28C. Deteremine the A260 value and the A260/A280 ratio for your prep.

Evaluating Yield, Purity and Size of the DNA in a Genomic Prep Before proceeding further into costly and time-consuming manipulations it is critical to analyze, at least in a cursory way, the quantity and quality of DNA in the prep. Estimating DNA Concentration by A260 The UV absorbance spectrum of DNA exhibits an Amax @ 260 nm based on the aromatic ring structures of the DNA bases. This is the most convenient way to estimate DNA concentration and calculate yield, as long as the DNA preparation is relatively free of contaminants that absorb in the UV. Proteins, and residual phenol left from the isolation procedure, are typical contaminants that may lead to an overestimate DNA concentration. An A260 = 1.0 indicates a [DNA] = 50 ug/ul, assuming the DNA is pure. Detecting protein contamination by A260/A280 Protein contaminants in a DNA prep also will absorb UV but their Amax = 280 nm. Therefore, the A260/A280 ratio can reveal the presence of gross amounts of protein contamination. Pure DNA has an A260/A280 ratio of 1.8-1.9. Lower ratios indicate substantial protein contamination. A higher ratio generally indicates RNA contamination.

Agarose Gel Electrophoresis Gel electrophoresis can confirm that the DNA in your prep is HMW and will reveal if there is a large amount of RNA contamination. We will analyze the genomic DNA prep and several other samples by gel electrophoresis in a later lab.

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Assignment 1. Make a rough calculation of the theoretical DNA yield in (in ug) and DNA concentration (in ng/ul) assuming that you recover 100% of the genomic DNA in pure form. You will need the following ballpark assumptions: 2 X 109 cells/ml 4,500 kb 616 g/mole 1 ml 0.1 ml 6.2 X 1023 molecules/mole Typical concentration of an overnight culture: Approximate Genome size Average MW of DNA base pair Original culture volume Final DNA sample volume Avogadro's #

2. Record the actual results of the analysis in the following format:

1 ml culture 2X10 9 cells/ml approx.

Genomic DNA Prep.

vol. = ____ ul conc. = _____ ng/ul amount = _____ ug yield = _____%

"yield" is the % of theoretical yield calculated in part 1. 3. Briefly comment on the quality of your DNA prep.

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