a. Principle :
DNA and
RNA exist in cell in combination with other macromolecules such as proteins and
carbohydrates. Cells are first lysed with enzyme proteinase and detergents such
as SDS or Sarkosyl or Triton X-100. These detergents dissolve the lipids, and
thus membranes, releasing the DNA and RNA out of the cell. EDTA is added to
chelate Mg++ required for nuclease
activity, which otherwise destroys the nucleic acid. Proteins which complex
with DNA and RNA are then removed by successive extraction with phenol.
Residual phenol must be subsequently removed completely since its traces are
inhibitory to most enzymes used in recombinant DNA techniques.
B.
Materials :
1) TE buffer (pH 8) (Appendix I)
2) 10% SDS
3) Proteinase K (Appendix III)
4) 5 M NaCl
5) CTAB/ (Cetyl trimethyl ammonium bromide)/
NaCl (Appendix III)
6) Chloroform
7) Isopropanol
8) Tris saturated phenol (Appendix III)
9) Ethanol
c. Method
:
1) Take
about 2 Lakhs of B. malayi microfilariae in 0.5 ml normal saline in an
eppendorf tube. Spin for 10
minutes at 6000 rpm at room temperature and remove the supernatant.
2) To the
Pellet add 567 µl of TE buffer, 30 µl 10 % SDS and 3 µl Proteinase K (20 mg /
ml Double distilled Water.). The solution will be watery & sticky.
3)
Incubate for 1 hour at 37° C
4) Add 100
µl of 5 M NaCl, and mix thoroughly.
5) Add 80
µl CTAB / NaCl. mix well, incubate at
65° C for 15 minute.
6) Add 0.4
ml Chloroform and 0.4 ml Tris saturated phenol.
7) Spin
for 10 minutes (12000 rpm at room temperature)
8) Take
upper aqueous layer and add 0.6 volume of Iso-propanol.
9) Keep
for precipitation at –20° C overnight.
10) Spin
for 30 minute (10,000 rpm at 4° C).
11) Remove
supernatant immediately but slowly with pipette.
12) Keep
the pellet in eppendorf tube for drying till the propanol is completely evaporated.
13)
Suspend the pellet in TE buffer (100µl in each) and allow it to dissolve.
14) Take
out 10µl sample and check for its purity on 0.8% Agarose gel.
a.
Principle:
Extracted
DNA can be visualized on Agarose Gel Electrophoresis. The DNA is simply loaded into the wells and
allowed to migrate towards the anode. All molecules should have similar charge/
mass ratio, the rate of migration depends simply on the molecular weight. The
bands can be visualized with the help of intercalating agent ethidium bromide
under uv light. Using known standards the molecular weight of each fragment can
be determined.
B.
Material:
1)Agarose
powder
2)Ethidium
bromide (Appendix I & III)
3)TAE
(Tris Acetate) buffer (Appendix I)
4)UV
transilluminator.
5)Loading
buffer (Appendix I)
C. Method:
1) For 1%
Agarose gel weigh 1 gm of Agarose and add it into 100 ml TAE buffer.
2) Heat
the slurry in a boiling water bath or in a microwave oven until the agarose
dissolves.
3) Cool
the solution to 37°C. Add ethidum
bromide to a final concentration of 0.5µg/ml.
4) Seal
the edges of a clean dry glass plate with autoclaved tape so as to form a mold.
5) Using a
Pasteur pipette, seal the edges of the mold with a small quantity of agarose
solution.
6) When
the seal is set, pour the rest of the agarose solution into the mold and
immediately clamp the comb, the teeth of which will form the sample wells, into
position near one end of the gel. Check to see that there is 0.5-1.0 mm of
agarose between the bottom of the teeth and the base of the gel, so that the
sample wells are completely seal.
7) After
the gel is completely set (30-45 minutes at room temperature), carefully remove
the comb and autoclaved tape and mount the gel in the electrophoresis tank.
8) Add
just enough electrophoresis buffer to cover the gel to a depth of about 1mm.
9) Samples
are mixed with loading buffer (Appendix) and are loaded into the slots of the
submerged gel. Usually loading buffer is made up as a 6 fold to 10 fold
concentrated solution, which is mixed with the sample and then slowly applied
to the gel using a disposable micropipette, an automatic micropipetter or if
you have a steady hand, a Pasteur pipette.
10)Electrophoresis
is carried out at voltage up to 60-70 volts for 1-1.5 hours.
11)After
the electrophoresis is over, the bands can be visualized under UV light.