A. Genomic DNA Isolation

8/2/2019 A. Genomic DNA Isolation

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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 molecularbiology; from in vivo toin vitro, if you prefer.

DNA was first isolated as long ago as 1869 by Friedrich Miescher while he was a postdoctoral student atthe University of Tbingen. Miesher obtained his first DNA, which he referred to it as nuclein, fromhuman leukocytes washed from pus-laden bandages amply supplied by surgical clinics in the timebefore antibiotics. He continued to study DNA as a professor at the University of Basel, but switchedfrom leukocytes to salmon sperm as his starting material. Meishers choice of starting material wasbased on the knowledge that leukocytes and sperm have large nuclei relative to cell size. DNA isolatedfrom salmon sperm and from (bovine) lymphocytes is still available commercially.

Molecular biologists distinguish genomic DNA isolation from plasmid DNA isolation. If you have takenthe Biology 20L class you have done plasmid DNA isolation from E. coliusing a procedure based on acommercial kit from Promega (Wizard Miniprep System). Plasmid DNA isolation is more demandingthan 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 companiessell reagent kits. Choosing the most appropriate method for a specific application demandsconsideration 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, areconsiderably more difficult than animal cells, because of their cell walls, and require specialattention.

Most lab strains ofE. coliare fairly straightforward, but a few E. colistrains produce high molecular

weight polysaccharides that co-purify with DNA. You need to look into the genotype of the E. colistrain to know whether you need special steps to eliminate this extraneous material. Inasmuch asourE. coliisolates 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 ahigh yield. Fortunately, E. coliis easy to grow, and PCR is effective with very small amounts of DNAsample, 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 haveisolated 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 theDNA 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 andby DNases.

Hydrodynamic shear is minimized by avoiding vigorous vortexing and pipetting of DNA solutions. Asimple 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 theprocedure. Occasionally DNases are introduced to the procedure as accidental contaminants of otherreagents, particularly RNase. Many investigators buy special "Molecular Biology" grade reagents thathave been certified "DNase-free" by the manufacturer. These are expensive. DNases present ascontaminants 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 relativelyhigh yield, and was formerly widely used. However, ultracentrifugation is very expensive because itrequires an instrument costing around $ 40,000. Additionally, it is inconvenient because thecentrifugation runs typically go many hours. So this method is now used mostly in situations where highyield and high purity are critical.

Many biotech companies sell kits with all the reagents necessary for genomic preps. Youneed 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 notsurprising 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, butexpensive, when you consider the cost of hazardous waste disposal. Our procedure does not usephenol/chloroform.

LYSIS

Cell walls and membranes must be broken to release the DNA and other intracellular components. Thisis usually accomplished with an appropriate combination of enzymes to digest the cell wall (usuallylysozyme) 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 fromthe 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 commontechnique of protein removal involves denaturation and extraction into an organic phase consisting ofphenol and chloroform.

Another widely used purification technique is to band the DNA in a CsCl density gradient usingultracentrifugation.


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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 transfer1.0 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 therotor 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 tubeas biohazard waste.

Carefully remove as much of the supernatant as you can without disturbing the cell pellet. Thepellet may be on the side of the tube, not squarely on the bottom. I use my P1000 set to 950ul.

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 anddenature proteins. You may notice that the cell suspension is not as turbid as the cell cultureyou 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 roomtemperature 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 gunkconsists 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 tubecontaining 600l of room temperature isopropanol (IPA).

Be sure that you dont suck up and transfer any of the grungy precipitate. I use my P1000 setto 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.


15. Carefully pour off the supernatant (do not pipette) and invert the tube on clean absorbentpaper to drain for 2-5 minutes. You want the paper to wick off the IPA that drains down andcollects 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.


18. Carefully pour off the ethanol supernatant (do not pipette) and invert the tube on cleanabsorbent paper to drain. You want the paper to wick off the ethanol that drains down andcollects 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 pelletcompletely dry. When all the EtOH is gone there should still be some water left hydrating theDNA.

20. Add 100l of DNA Rehydration Solution (RH) to the tube and rehydrate the DNA by incubatingat 65C for 1 hour.

After 30 minutes, flick the bottom of the tube gently to facilitate dissolution and mixing.


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Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature orat 4C, preferably on a low speed shaker.

21. Analyze the DNA prep. by spectrophotometry using the Nanodrop spectrophotometer and thenstore 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, atleast 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 andcalculate yield, as long as the DNA preparation is relatively free of contaminants that absorb in theUV. Proteins, and residual phenol left from the isolation procedure, are typical contaminants thatmay 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, theA260/A280 ratio can reveal the presence of gross amounts of protein contamination. Pure DNA hasan A260/A280 ratio of 1.8-1.9. Lower ratios indicate substantial protein contamination. A higherratio 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 alarge amount of RNA contamination. We will analyze the genomic DNA prep and several othersamples 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 (inng/ul) assuming that you recover 100% of the genomic DNA in pure form. You will need thefollowing ballpark assumptions:

2 X 109 cells/ml Typical concentration of an overnight culture:4,500 kb Approximate Genome size616 g/mole Average MW of DNA base pair1 ml Original culture volume0.1 ml Final DNA sample volume6.2 X 1023 molecules/mole Avogadro's #

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

1 ml culture

2X109 cells/ml approx.

vol. = ____ ul

conc. = _____ ng/ul

amount = _____ ug

yield = _____%

GenomicDNA Prep.

"yield" is the % of theoretical yield calculated in part 1.

3. Briefly comment on the quality of your DNA prep.

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A. Genomic DNA Isolation