Protocol for DNA Isolation from plant sample

Using Neem(Azadirachta Indica) leaves

1. Pluck young ,healthy neem leaves.Wash them with tap water.Remove the midrib.wash
the leaf fragments with distilled water,then dry on tissue paper.
2. Weigh 1 gram of the leaf fragments.Transfer the leaves to a mortar and pestle.Add 5 ml
of pre-heated DNA Extraction Buffer(CTAB Buffer).Grind the leaves in the buffer finely to
a slurry. [Heating CTAB buffer:- Take 50 ml of CTAB Buffer in a test tube and heat it to
60-65 oC in a water bath.]
3. After the leaves are fully grinded,transfer 0.5 ml of the slurry into microcentrifuge
tubes,and incubate at 60 oC in a water bath for 1 hour.
4. Add equal volume(0.5 ml) of chloroform/isoamyl alcohol into the tubes.Mix gently for
10-15 minutes.Centrifuge at 10000 rpm for 10 minutes.
5. You will notice that there are 3 layers in the centrifuge tube.The lowest one consists of
cell debris.The middle layer consists of proteins.The topmost layer contains
DNA.Carefully,without disturbing the layers,transfer the topmost layer into a fresh
microcentfuge tube.
6. Add approximately equal amount of chilled isopropanol to the tube,keep it at room
temperature for 10-15 minutes.Centrifuge at 10000 rpm for 10 minutes.Discard the
supernatant carefully.
7. Add 0.5 ml of wash buffer(70 % ethanol) and mix the tube by tapping and
inversion.Then centrifuge the tube at 10000 rpm for 10 minutes.Discard the
supernatant and allow ethanol to evaporate by keeping the tubes open for 10-15
minutes.
8. Resuspend the pellets in 50 l of TE Buffer.Store at 4 C for further use.
Equipments and Materials required:

Centrifuge
Mortar and pestle
Water bath
Micropipette(10-100 l , 100-1000 l)
Microcentrifuge tubes
Pipette tips (0.2-10 l ,10-100 l , 100-1000 l)
Tissue Paper

Chemicals required:1. CTAB Buffer[DNA Extraction Buffer]

2 % CTAB(Cetyl Trimethyl Ammonium Bromide)
20 mM EDTA
100 mM Tris HCl pH 8.0
1.4 M NaCl
Adjust pH to 7.5-8.0 and autoclave
2. Wash Buffer(70 % Ethanol)
3. Chloroform:Isoamyl Alcohol(24 : 1)
4. Isopropanol
5. TE Buffer
10 mM Tris Base
1 mM EDTA
pH 8.3

Protocol for Agarose Gel Electrophoresis

1. Wipe clean the gel comb,gel casting tray with 70 % Ethanol soaked in filter paper.Set
the gel combs in the gel casting tray.
2. Weigh agarose powder according to desired concentration of gel.(Usually 1 % or 1.5 %
is taken)
3. Pour the agarose in 1 X TAE Buffer.Heat the agarose till it fully dissolves in the buffer.
4. Wait for some time till the temperature of the gel becomes nearly equal to the body
temperature so that when u touch it,it does not scald your skin.Then add Ethidium
Bromide(5 l of EtBr for 100 ml of gel.The concentration of EtBr is 10 mg/ml).And mix it
thoroughly.
5. Pour the gel carefully into the gel casting tray.Wait for the gel to solidify.
6. Then carefully remove the gel combs and transfer the gel into the gel tank.
7. Pour 1 X TAE Buffer into the gel tank till the gel is completely submerged in the tank.
8. On a clean surface,mix 6X gel loading dye and DNA sample according to the formula:Volume of 6X gel loading dye = Volume of DNA Sample/6
{Example:If you want to load 10 l of DNA ,then you must mix 10/6 = 1.67 l of 6X Gel loading
dye fully with the DNA and load into the wells.}
9. Carefully load the sample(DNA + Gel loading dye) into the wells.
10.Cover the tank and connect the tank to electrophoresis power supply unit,at 70 V.
11.Observe the gel Using a UV-Transilluminator or a Gel documentation system.
Equipments and Materials required: Gel casting tray
Gel combs
Gel tank
Electrophoresis power supply
UV Transilluminator/Gel documentation system
Micropipette(0.2-10 l)
Chemicals Required:

Ethidium Bromide(10 mg/ml)

1 X TAE Buffer(Can be prepared from 50 X TAE Buffer)
50 X TAE Buffer composition:- (For 1 litre solution, 242 g Tris Base, 57.1 ml Glacial
Acetic acid, 100ml of 0.5 M EDTA, pH 8.3)
6X Gel loading Dye[ 0.25 % Xylene-Cyanol(W/V) , 0.25 % Bromophenol Blue(W/V),
30% Glycerol(V/V) ]