Download Isolation and Purification of Total Genomic DNA from Gram

10/22/2012
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 student will prepare 2 DNA samples from the Pseudomonas strain that was isolated from soil.
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. (Remember that you are doing
this in duplicate.)
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 950980 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
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 (1 kb = 1,000 base pairs)
Average MW of DNA base pair
Original culture volume
Final DNA sample volume
Avogadro's #
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