Do you know how to extract DNA ? - The Veterinary Site

Do you know how to extract DNA ?


DNA:-
(by MAAZ AHMED SIDDIQUI)
·         Stands for Deoxyribonucleic acid
·         Is a molecule that carries the genetic instructions used in the growth, development, functioning and reproduction
·         Of all known living organisms.
DNA STRUCTURE:
·         is made up of molecules called nucleotides
·         each nucleotide contains:
      -phosphate group
      -sugar group
      -nitrogenous base
·         Four types of bases are:
    -Adenine (A)                        -Thymine (T)
   -Guanine (G)                         -Cytosine (C)
·         The order of these bases is what determines genetic code or DNA’s instruction
·         The order of nitrogen bases in a DNA sequence form genes which tells cells how to make proteins.
·         DNA exists as a double helix structure with a sugar phosphate backbone.
DNA EXTRACTION:
·         Is the technique used to isolate DNA in a biological sample.

Importance:

·         Crime Identification

·         Historical Identification

·         Studying the genetic causes of various kinds of diseases.
·         For the development of diagnostics and drugs
·         Sequencing genome
·         Determination of the paternity







DNA EXTRACTION PROTOCOL:
1.      Take 600 μl of CLB (cell lysis buffer) in pre-labeled Eppendorf tube.
2.      Add 300 μl of EDTA/heparin blood and mix gently by inverting 4-6 times.
3.      Centrifuge at 4000 rpm for 5 min.
4.      Decant the supernatant and wash the pallet again with CLB 400 μl.
5.      Centrifuge at 5000 rpm for 5 min.
6.      Repeat washing (3-4 times) until all the hemoglobin is washed out.
7.      Add 450 μl NLB (Nuclear Lysis Buffer) and 100 μl of saturated NaCl to dried pallet.
8.      Add 550 μl pre chilled chloroform. Centrifuge at 5000 rpm for 3 min. Two separate layers will be formed.
9.      Withdraw the supernatant without touching the central layer to new labeled Eppendorf tube. This supernatant will be processed for further DNA extraction.
10.  Add 1000 μl pre-chilled ethanol.
11.  Place tubes at -20°C for 30 min and then centrifuge at 14000rpm for 6 min and discard ethanol slowly.
12.  Add 1000 μl of 70% ethanol.
13.  Centrifuge at 14000rpm for 6 min. Decant ethanol and let tube dry completely.
14.  Add 100 μl of T.E buffer and store at 37°C for 24hrs.
15.  After that check DNA on 0.8% agarose gel and quantify using NanoDrop.
16.  Store DNA at-20°C.


                                  DNA EXTRACTION BUFFERS
·         CELL LYSIS BUFFER:
1.      Tris HCl            100ml
2.      Mgcl2                1.01g
3.      KCl                    0.521g
4.      Sucrose              109.54g
5.      TRITON X100  10ml
6.      Dist. H2O          make volume up to 1000

·         NUCLEAR LYSIS BUFFER:
1.      Tris HCl          100ml
2.      Sodium citrate 2.9g
3.      EDTA              20ml
4.      SDS                  20g
5.      Dist. H2O         make volume up to 1000

·         T.E BUFFER:
1.      Tris HCl          10ml
2.      EDTA              2ml
3.      Dist. H2O        make volume up to 1000

·         NACL SOLUTION:
1.      350 g in 1000ml

·         TRIS HCL:
1.   Tris                  121.1g
               2.   Dist. H2O        make volume up to 1000
               3.   HCl                  make pH 7.6
·         EDTA SOLUTION:
               1.  EDTA               186.1g
2.      Dist. H2O         make volume up to 1000
3.      NaOH               make pH 8

                    What each reagent is for?
1.     Lysis buffer :
·         lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments
            -CLB:
¨      Breaking the cell membranes open to expose the DNA along with the cytoplasm within the cell
      -NLB:
¨      Breaking the nucleus

2.     Triton X-100 and SDS :
·         Detergents
·         Are added to break up membrane structures.

3.      EDTA :
·         EDTA is responsible for chelation of divalent ions
·         EDTA helps to stop DNases (DNA cutting enzymes) present in the cytoplasm from acting on the exposed DNA
·         Mg2+ is an important factor for activity of DNases. EDTA deprives the enzyme of this co-factor and renders it inactive
·         DNases are enzymes that “chew up” DNA and thus reduces the yield of genomic DNA. So it’s important to keep them from acting on DNA of interest.

4.     Tris-HCl:
·         Maintains pH

5.      Salt solution :
·         The solution is treated with salt solution to make debris such as broken proteins, lipids and RNA to clump together.
·         Sodium chloride helps to remove proteins that are bound to the DNA.

6.     CHLOROFORM :
·         Used to help separate proteins, lipids and polysaccharides from nucleic acids in the cell
·         Chloroform and water separates into two distinct phases. Lower phase will be chloroform containing proteins ,lipids and polysaccharides

7.      Chilled ethanol :
·         DNA doesn’t dissolve in ethanol. It will aggregate together, giving a pellet upon centrifugation
·         Used to separate pure DNA
·         Colder temperature reduces activity of enzymes that can break down DNA.

8.      70% ethanol:

·         This step is to wash any residual salt away from the pelleted DNA.

9.      TE buffer :
·         Used to solubilize DNA while protecting it from degradation


Do you know how to extract DNA ? Do you know how to extract DNA ? Reviewed by Maaz ahmed siddiqui on 10 May Rating: 5

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