Docstoc

DNA Extraction Introduction Why do people want to obtain genomic

Document Sample
DNA Extraction Introduction Why do people want to obtain genomic Powered By Docstoc
					Hana Hakami                                                           2011


                           DNA Extraction
Introduction

       Why do people want to obtain genomic DNA? There are a number
of reasons. For example, this DNA could be used to clone new genes or
look for special regions of interest. You could also obtain a number of
different genomic DNA and make comparisons with them to identify
genetic diseases.

      The first step in obtaining DNA involves breaking open the cell’s
membrane by using physical or chemical means. For example, you could
use ultrasonic waves or vibrations (sonification). Alternatively, our
method involves homogenization using a mortal and pestle and
detergent to disrupt the cells’ membrane.

Procedure for DNA extraction

      DNA will be extracted from whole blood using Traditional Method
(John M. S. Bartlett and Anne White).

      There are many differing protocols and a large number of
commercially available kits used for the extraction of DNA from whole
blood. This procedure is one is used routinely in both research and
clinical service provision and is cheap and robust. It can also be applied
to cell pellets from dispersed tissues or cell cultures (omitting the red
blood lysis step.

Principle

      Most mammalian whole blood and bone marrow specimens
contain both non-nucleated cells (red blood cells) and nucleated cells
(white blood cells) which contain DNA.

       When purifying DNA from these whole blood or bone marrow
specimens, the red blood cells, which lack genomic DNA, are first lysed
to facilitate their separation from the white blood cells.
Hana Hakami                                                           2011


       DNA is purified from cells such as white blood cells, animal tissue,
cells contained in body fluids, or microbes, by first lysing the cells with
an anionic detergent in the presence of a DNA stabilizer.

       The DNA stabilizer works by limiting the activity of DNases that
are contained in the cell and elsewhere in the environment.
Contaminating RNA is then removed by treatment with an RNA digesting
enzyme. Other contaminants, such as proteins, are removed by salt
precipitation. Finally, the genomic DNA is recovered by precipitation
with alcohol and dissolved in a buffered solution containing a DNA
stabilizer.

Materials

Glass wares - 15 ml Centrifuge tubes - 1.5 ml Microfuge tubes - Pasteur
pipettes - Tissues - Gloves – Biohazard Bags & Container

Equipments

Centrifuge – Waterbath set at 65⁰C - roller/rotator - Vortex – pH meter -
Freezer (-20⁰C) – Sensitive Balance – Ice Maker

Chemicals

Tris base - EDTA - Sodium Per-chlorate - Sucrose - MgCl2 - NaCl -
Chloroform - Ethanol - SDS (Sodium Dodecyl Sulphate) - Trition x-100 -
HCl - NaOH - Clorox

Reagents

      1. Tris-HCl (1 M) pH7.6 : 121.1g Tris base - 800 ml d.H2O 
         Adjustment pH to 7.6 by HCl.
      2. EDTA (0.5 M) pH 8.0 : 146.1 g EDTA - 800 ml d.H2O 
         Adjustment pH to 8.0 by NaOH  Make up to 1 L d.H2O.
      3. Sodium Per-chlorate (5 M) : 70 g Sodium Per-chlorate - 80 ml
         d.H2O  Make up to 100 ml d.H2O.

      *You can prepare 5 M Sodium Perhclorate by use Sodium Chloride
      (29.250g NaCl wet in 100 ml d.H2O).
Hana Hakami                                                          2011


       4. Reagent A (RBC Lysis Solution): 10 ml 1M Tris-HCl (pH 7.6)–
          109.54 g Sucrose - 0.47 g MgCl2 - 10 ml Trition x-100  Make
          up to 800 ml d.H2O  Adjustment pH to 8.0 Make up to 1 L
          d.H2O.
       5. Reagent b (Cell Lysis Solution) : 400 ml 1M Tris-HCl (pH 7.6)/
          120 ml 0.5 M EDTA (pH 8.0) - 8.76 g NaCl  Adjustment pH to
          8.0  Make up to 1 L d.H2O  10 g SDS.
       6. TE Buffer pH 7.6 (DNA Hydration Solution): 10 ml 1M Tris-HCl
          (pH 7.6) - 2ml 0.5 M EDTA (pH 8.0)  Make up to 1 L d.H2O 
          Adjustment pH to 7.6.

Protocol

1st: RBC Lysis

       1.   Prepare 3 ml of whole blood sample in EDTA tube.
       2.   Transfer the blood sample to 15 ml tube.
       3.   Add 12 ml Reagent A.
       4.   Mix on rolling for 4 min at room temperature.
       5.   Centrifuge at 3000 g for 5 min at room temperature.
       6.   Discard supernatant gently .
       7.   Remove moisture by inverting the tube on tissue paper.

2nd: Cell Lysis

       1.   Add 1 ml Reagent B on cell pellet.
       2.   Vortex briefly to re-suspend the cell pellet.
       3.   Add 250 ml 5 M sodium per-chlorate.
       4.   Mix by inverting tube several times.
       5.   Place tube in water bath for 15- 20 min at 65⁰C.
       6.   Cool to room temp.

3rd: Protein Precipitation

       1.   Add 2 ml ice-cold chloroform.
       2.   Mix on a rolling or rotating mixer for 30-60 min.
       3.   Centrifuge at 2500g for 5 min.
       4.   Transfer upper phase into a clean 15 ml tube using a sterile
            pipette.
Hana Hakami                                                              2011


4th: DNA Precipitation

      1. Add 2 to 3 ml ice- cold ethanol gently on tube wall and invert
         gently to allow DNA to precipitate.
      2. Centrifuge at 2500g for 3 min.
      3. Discard from supernatant gently.
      4. Allow to air dry the DNA pellet by invert the tube on
         absorption tissue for 10-20 min .

5th: DNA Hydration

      1. Re-suspend the pellet in 100-300 µl of TE buffer.
      2. Rehydrate DNA by incubating at 65°C for 1 hour and leave it
         overnight at room temperature.
      3. Centrifuge the DNA sample briefly by short spin (Spin-down).
      4. Transfer the DNA sample to 1.5-ml Eppendorf tube (microfuge
         tube)

      * Do not forget fill full information about DNA sample on
      Eppendorf tube (name, type of sample and date ).

      * As a final step in nucleic acid isolation, the yield and purity of the
      extracted nucleic acid may need to be determined.

6th: Storage of DNA

       Original stock of each sample of DNA will be labeled and stored
        at -80°C.
       From each sample 100 µl working DNA (for example; 50ng/µl
        for all working DNA samples) will be labeled and stored at 4°C
        or -20° C.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:9
posted:8/19/2012
language:English
pages:4