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Isolation and Purification of Total Genomic DNA from Gram-Negative Bacteria

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, as it were. DNA was first isolated as long ago as 1869 by Friedrich Miescher while he was a postdoctoral student at the University of Tübingen. 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. Meisher’s 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. Plasmid DNA isolation from E. coli (the plasmid "miniperep") is such a routine procedure that it is often done in high school biology labs. Plasmid DNA isolation is more demanding than genomic DNA isolation because plasmid DNA must be separated from chromosomal DNA. In a genomic DNA isolation you need only need 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.

This procedure works well with most Gram-negative bacteria including most lab strains of E. coli. 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.

• YIELD: How much DNA do you need? Consider your downstream application/s for the isolated DNA. 1-2 ml of cell culture can provide enough DNA for analysis by gel electrophoresis, for PCR, and for a couple restriction digests.

• 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.

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• INTEGRITY: How large are the DNA fragments in our genomic preps?

Very high molecular weight (HMW) DNA is fragile and may be cut into smaller fragments, or degraded altogether, by hydrodynamic shearing and by DNases.

Hydrodynamic shear is minimized by avoiding vigorous and repeated 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 DNA isolation 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 DNA preps. You need to look carefully at the cost of these kits relative to the labor that they save.

• SAFETY

DNA isolation methods are designed to break cells and denature proteins, iso t is not surprising that some reasonably nasty reagents are involved.

The 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 80˚C 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 HMW DNA on the basis of their higher solubility 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.

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GENERAL DIRECTIONS Each pair of students will prepare 2 DNA samples from the same strain that was used for the P1 lysogen construction. ("L-1", etc.) The 2 preps will be identical except that the RNase treatment will be omitted for one. 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 950-980 ul.

4. Resuspend the cell pellet in 600µl 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 80°C 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 3µl of RNase solution to the cell lysate. Invert the tube 2–5 times to mix. RNase will be dispensed from the front bench. Omit the RNAase for ONE of the two samples.

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8. Incubate at 37°C 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 µl of Protein Precipitation Solution (PPS) to each tube.

Vortex vigorously at high speed for 20 seconds. Do not skimp on the vortexing 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 µl) containing the DNA to a clean 1.5ml microcentrifuge tube

containing 600µl of room temperature isopropanol (IPA). Be sure that you don’t 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 600µl 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 10–15 minutes.

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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 100µl of DNA Rehydration Solution (RH) to the tube and rehydrate the DNA by incubating

at the tube overnight at room temperature on a low speed shaker. Be sure that you tube is clearly labeled before leaving it on the shaker.

Assignment Make a rough calculation of the theoretical total 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 Typical concentration of an overnight culture: 4,500 kb Approximate Genome size (1 kb = 1,000 base pairs) 616 g/mole Average MW of DNA base pair 1 ml Original culture volume 0.1 ml Final DNA sample volume 6.2 X 1023 molecules/mole Avogadro's #