Docstoc

Isolation and Purification of Total Genomic DNA from coli

Document Sample
Isolation and Purification of Total Genomic DNA from coli Powered By Docstoc
					                                                                                          2/19/2010


             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 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. 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.




                                                                                              1 of 6
                                                                                        2/19/2010


• 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 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 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.




                                                                                             2 of 6
                                                                                      2/19/2010


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 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.

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 the RNase-treated cell lysate.

     Vortex vigorously at high speed for 20 seconds. Do not skimp on the vortexing



                                                                                           3 of 6
                                                                                    2/19/2010


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.

   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 65°C for 1 hour.

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


                                                                                        4 of 6
                                                                                     2/19/2010




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

21. Analyze the DNA prep. by spectrophotometry using the Nanodrop spectrophotometer and then
    store the DNA at 2–8°C.

   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.




                                                                                         5 of 6
                                                                                     2/19/2010


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                  Typical concentration of an overnight culture:
     4,500 kb                          Approximate Genome size
     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 #

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


                                                               vol. = ____ ul
                                             Genomic
                  1 ml culture               DNA Prep.         conc. = _____ ng/ul

                  2X10 9 cells/ml approx.                      amount = _____ ug

                                                               yield = _____%



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

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




                                                                                        6 of 6

				
DOCUMENT INFO