Viola CTAB Extraction gel by benbenzhou

VIEWS: 79 PAGES: 6

More Info
									Laboratory 1. genomic DNA Extraction and Gel Electrophoresis

Exercise 1. DNA Extraction using CTAB:


              Materials:
              CTAB solution                 β-mercaptoethanol
              Liquid nitrogen               3M NaOAc
              Phenol/EB pH 8.0              100% isopropanol
              96:4 (chloroform: Isoamyl)    70% EtOH
              Elution Buffer (EB)




   1. Pre heat 500μl CTAB in 65ºC water bath.
   2. Place 0.2 to 0.5g plant material into the mortar and add liquid nitrogen, grind to a
       powder.
   3. Add plant material to the pre-heated CTAB and add1%β-mercaptoethanol (5μl) to
       the centrifuge tube and incubate at 65˚C for 0.5 hr. Vortex well at least every
       10min.
   4. Centrifuge for 8min. at 13,000rpm, transfer top layer to new microcentrifuge tube.
   5. Add 500ul 96:4 chlorofor:isoamyl alcohol, vortex well, centrifuge for 3min.,
       transfer top layer to a new microcentrifuge tube.
   6. Repeat step 5.
   7. Add 0.1 volume 3M NaOAc and 0.7 volumes 100% isopropanol, vortex,
       incubate in ice bath for 10min. Centrifuge for 10min. at 13,000 and discard
       supernatant. Be careful not to disturb the pellet.
   8. Wash pellet in 500μl cold 70% EtOH, invert 5X; incubate 10min. in an ice bath.
   9. Centrifuge for 2min. add maximum speed to secure the pellet, discard the
       supernatant.
   10. Dry the pellet by placing inverted, cap open, in the 37o incubator for 10 min.
   11. Add 50μl EB Buffer, store in 4˚C overnight before quantifying.

               CTAB Solution:
                   2g CTAB
                   2g PVP
                   28ml 5M NaCl (29.22g/100ml)
                   4ml EDTA pH 8.0
                   10ml 100mM TrisHCl pH8.0
                   1% β-mercaptoethanol (just before use)

              ·Prepare the solution without CTAB, PVP, β-mercaptoethanol and
              autoclave for 20 min.
              ·After autoclave add CTAB and PVP; heat to dissolve CTAB and PVP on
              the hot tray. Add the 1% β-mercaptoethanol directly to the extraction
              vial (5μl/500μl of CTAB solution). Pre-heat the solution in a 65˚C water
              bath before use.
              General Considerations and Rationale
Three components are necessary for successful nucleic acid extraction: 1) inhibition of
nucleases, 2) removal of proteins, and 3) physical separation of the nucleic acid from
other cellular components. Nuclease inhibition and removal of proteins are not mutually
exclusive and often are accomplished in a single step (such as phenol extraction).

Inhibition of Nucleases

Detergents inhibit nucleases and help separate the proteins from the nucleic acids. A
common detergent is sodium dodecylsulfate (SDS). Avoid the use of potassium salts or
temperatures below 10 °C with SDS since this may cause precipitation of the detergent.
One of the most widely used detergents for plant DNA extraction buffers is CTAB
(cetyltrimethyl-ammonium bromide). CTAB is most often used in a 2% (w/v) solution
(Rogers & Bendich 1985, Doyle & Doyle 1987, Nickrent 1994).

Removal of Proteins

Phenol is a strong denaturing agent for proteins. In phenol extractions, proteins partition
into the organic phase (and interface) whereas nucleic acids partition in the aqueous
phase. Usually phenol is used in a 1: 1 mixture with chloroform since deproteinization is
more effective when two different organic solvents are used simultaneously. In addition
to denaturing proteins, chloroform is useful in removing lipids and a final chloroform
extraction helps to remove the last traces of phenol. The isoamyl alcohol helps with the
phase separation, decreases the amount of material found at the aqueous and organic
interface, and helps reduce foaming.

Antioxidants such as 8-hydroxyquinoline or ß-mercaptoethanol are often added to
phenol. During phenol extractions, the pH of the buffer is important in determining
whether DNA and/or RNA are recovered. At pH 5-6, DNA is selectively retained in the
organic phase leaving RNA in the aqueous phase (hence water saturated phenol is useful
for RNA extractions). At pH 8.0 or higher, both DNA and RNA are retained in the
aqueous phase. Phenol can be stored under buffer for up to one month.

Protease

The most common proteolytic enzyme used in nucleic acid extractions is protease K (e.g.
Sigma P4755) which is generally made up in 50 units/ml aliquots and stored frozen for
immediate use during extractions. Solutions are generally incubated with this enzyme at
37 °C for at least one hour.

Other Extraction Buffer Components

Two common reducing agents found in extraction buffers are ß-mercaptoethanol or
dithiothreitol (DTT). EDTA is also present to chelate Mg+2 ions thus mediating
aggregation of nucleic acids to each other and to proteins.
Ethanol and/or Isopropanol Precipitation

The most common method of concentrating nucleic acids is with alcohol precipitation.
This occurs in the presence of a salt (see below) at low temperatures (-20 °C or less).
Either 2.0 volumes of ethanol can be used to precipitate DNA or 0.6 volumes of
isopropanol. Isopropanol is often used for the first precipitation, but not for final ones
because it tends to bring down salts more readily than ethanol. Isopropanol can also be
used when the volume of the tube will not allow the addition of 2 volumes of ethanol.
The nucleic acid is collected by centrifugation at 10,000 rpm for ca. 20 - 30 minutes.

The most common monovalent cations used in nucleic acid precipitations are shown in
the following table:

                      Stock Solution              Final Concentration
Sodium acetate        2.5 M (pH 5.2 - 5.5)        0.25 M to 0.3 M
                                                  0.10 M (optimal < 0. 15 M = 1/50
Sodium chloride       5.0 M
                                                 vol.)
 Ammonium
                      4.0 M                      1.3 M
acetate*

* Filter sterilize, do not autoclave

The choice of salt is determined by the nature of the sample and the intended use of the
nucleic acid. Samples with phosphate or greater than 10 mM EDTA should not be
ethanol precipitated because the salts will come down with the nucleic acid.

Butanol Extractions

DNA is recovered from dilute solutions by extracting with 2-butanol. The water from the
sample moves into the butanol which is discarded, thus leaving a higher DNA
concentration in the aqueous phase. Water saturated butanol is also used to remove
residual ethidium bromide from samples obtained via CsCl centrifugation or agarose gel
purification.

Reagents:

         2X CTAB buffer: 100 mM Tris-HCI, 1.4 M NaCl, 30 mM EDTA, and 2 %
        (w/v) CTAB. The buffer is usually ca. pH 8.0 without adjustment.
         protease K (Sigma Type H, ca. 0. 13 units/mg, P-4755). I store the enzyme in
        50 units/1 ml aliquots in the freezer.
         Dithiothreitol (DTT) 0.5 M. Stored as 0.5 ml aliquots - added to buffer just
        before use.
         1X TE buffer
         Ammonium acetate, 4.0 M.
         Organics: chloroform:isoamyl alcohol (24: 1), Tris buffered phenol, cold
        isopropanol, cold 100% ethanol, and 70% ethanol.

The best DNA extracts come from young plant material. Attempt to conduct the
extraction soon after collection if refrigeration is not possible.
Day 2
 Exercise 2. Gel Electrophoresis

       The standard method for separating DNA fragments is electrophoresis through
agarose gels. DNA is applied to a slab of gelled agarose and then an electric current is
applied across the gel. Because DNA is negatively charged (phosphate groups in the
backbone), it migrates through the gel towards the positive electrode. The rate of
migration depends on:

1) the size of the fragment; the smaller the DNA, the faster it "runs" through the gel;

2) the concentration of agarose; the higher the concentration, the more the agarose retards
the movement of the DNA fragments; and

3) the voltage applied to the gel; the higher the voltage the quicker the DNA "runs" (but
at a trade-off--the DNA fragments do not separate as efficiently).


a. Pouring the gel

A demonstration on preparing and running an agarose gel will be given. Each group
should have a gel apparatus and cover, two end walls, two combs and a plastic gel tray.
The gel tray should be placed in the chamber and the two end walls inserted. A comb
should be placed near one of the end walls.

Make 100 ml of a 1.0% agarose in 1X TBE for your gel in a 250 ml flask. Minigels
require ~30 ml agarose.

Use the microwave to melt the agarose. Be sure the solution is completely melted and
homogenous.

Warning!! Never leave the agarose solution unattended when using the microwave. The
solution must be frequently swirled during the heating process to prevent superheating of
local areas. Always use "hot hands" or autoclave gloves when heating the agarose.


You will be provided Ethidium Bromide (EtBr) at a concentration of 1 mg/ml in a 1.5
microfuge tube. This tube should be saved and stored at 4ºC.

Warning!! Ethidium bromide is a mutagen. Remember to wear gloves when working
with EtBr and to dispose of contaminated tips in specially marked containers. Gels
should be placed in a plastic bag for disposal. Microwaving EtBr produces harmful
volatiles.


Add EtBr to a final concentration of 0.1 to 0.5 g/ml to agarose immediately before you
pour the gel. To do this, label a 50 ml conical tube EtBr/agarose. Add the EtBr to the
tube and then pour about 30 mls of the agarose into the tube. Cap and invert a few
times. The agarose solution should be cooled to about 50ºC (a temperature at which one
can hold the flask, but it is uncomfortable) before pouring the gel. Pouring the gel when
the agarose is too hot may damage the gel apparatus.

Pour the 1.0% agarose minigel with 0.1 g/ml EtBr and allow it to solidify.

Practice loading a few lanes with loading dye if you've never loaded a gel before. This
can be run into the gel without affecting the migration of DNA added to the well at a later
time.

At first it will be easier to load the wells dry. Add 1X TBE buffer to submerge the gel
prior to electrophoresis. Later, you should become adept at loading wells submerged in
buffer.


b. Loading the Samples and Running the Gel:

To a new eppendorf or on parafilm, add 5 l loading dye to 15 l of your sample and
finger flick. Load 20 l total volume of your samples per well for minigels with 8-well
combs. Load 10 l of the molecular weight markers provided. Your gel should contain:

1. molecular weight markers
2. gDNA group 1
3. gDNA group 2
4. gDNA group 3

Run the gel at 100 V until the bromophenol blue is about 3/4 through the gel (approx. 1 h).

Using gloves, carefully remove the tray with the agarose gel. Take it to the UV
transilluminator and slide the gel off the tray onto the lamp. Wearing UV-protective
goggles (NOT YOUR SAFETY GLASSES), examine the gel. The cover of the
transilluminator may also be used to protect your eyes. Photograph your gel with a ruler
for your notebook.
Exercise 3. Quantifying DNA Using a Bio Photometer (Spectrophotometer)
              (may be done at the second class)

   -   turn the BioPhotometer on and allow to warm up (switch on back left)
   -   choose the correct wavelength by pressing the appropriate button (dsDNA)
   -   Put 49 l of EB buffer into a clean cuvette (be careful to touch only the frosted
       sides of the cuvette)
   -   Insert the cuvette into the port and “blank” the machine
   -   Remove the cuvette and add 1 l of you DNA
   -   Record the readings


                                                       BioPhotometer Display Readout

                                                               A)                    g/ml


                                                  B)           260/280
Detection of DNA and RNA with Absorption
Spectroscopy.                                     C)           260/230



A - This number gives you the concentration of nucleic acids (DNA and RNA) measured
as micrograms per milliliter (g/ml)

Values B and C give you an idea of how clean your sample is.

   B - The ratio of the absorbencies of the sample at two discreet wavelengths of light –
       260 nanometers (nm) and 280 nm. Nucleic acids have a peak absorption at 260
       nanometers while proteins have a peak absorption at 280 nm. Protein
       contamination will reduce the 260/280 ratios.

   C - The ratio of the absorbencies of the sample at two discreet wavelengths of light –
       260 nanometers (nm) and 230 nm. Again, nucleic acids have peak absorption at
       260 nanometers while contaminants containing peptide bonds and phenols have
       peak absorption at 230 nm. Relatively higher contaminant concentrations will
       reduce the 260/230 ratios.

								
To top