Agarose gel electrophoresis are performed generally on the
horizontal slab gels, which very easy to handle and has the
advantages of simplicity in pouring, loading and handling,
versatility in size using the same apparatus. Tanks are available in
all sizes and can be cheaply made according to a required design.
The electrophoresis is carried out in a buffer which is either Tris-acetate
EDTA (TAE) or Tris-borate EDTA (TBE). Tris-acetate has a low
buffering capacity and it tends to become exhausted during extended
electrophoresis (the anode becomes alkaline, the cathode becomes
acidic) whereas Tris-borate has higher buffering capacity. Tris-borate
gives better resolution than Tris-acetate.
Materials and Reagents
Agarose (as per the requirement, see table below)
1x Tris-Borate EDTA buffer pH 8.0/ 1x Tris-Acetate
EDTA buffer pH 8.0
10 x loading buffer
λ 1 Kb ladder
Composition of reaction mixtures
A. Agarose (May change
according to the electrophoresis unit)
Gel size (cm)
1x Gel buffer
11 × 14
11 × 20
20 × 20
20 × 25
50 µl (thick comb)
B. 50x TAE (Tris-Acetate
EDTA) buffer pH 8.0 ( 1 litre)
242 g Tris base in 750 m ddH2O
57.1 ml Glacial acetic acid
100 ml 0.5M EDTA pH 8.0
Stir, do not adjust the pH
Make up to 1000 ml
Dilute to 1x concentration for the use in electrophoresis by taking
40ml of 50x buffer and making the volume to 2000ml by adding 1960 ml
C. 5x TBE (Tris-Borate
EDTA) buffer pH 8.0 (1 litre)
54 g Tris base
27.5 g Boric acid
20 ml 0.5M EDTA, pH 8.0
Stir, do not adjust the pH
Sterile distilled water to 1000 ml
Dilute to working concentration of 1x by taking 400 ml of 5x TBE and
diluting it to 2000 ml using 1600 ml of ddH2O
D. Ethidium bromide
10 mg/ ml
Ethidium bromide is highly carcinogenic.
Always wear gloves and protective goggles while
handling ethidium bromide.
Do not dispose to open environment.
E. 10x loading buffer
1.6 g of bromophenol blue (40%)
0.16 g of xylenecyanol FF
Mix with 50 ml glycerol. Make the volume up to 100 ml.
Aliquot to 1.5 ml tubes.
Heat the tube for 10 minutes, cool and store at 4°c.
F. Hind III
1. Digest 200 µl
of λDNA (0.25 µg/µl)
using 25 µl of HindIII
enzyme. Incubate the reaction at 37°c overnight.
2. Test the DNA for complete digestion. If the DNA is digested
incubate the mixture at 65°c for 10 minutes.
3. Distribute the 250 µl to five 1.5 ml tubes
each to have 50 µl and make up the volume of each 50 µl to 200 µl by
adding 20 µl of loading buffer and 130 µl of distilled water to make
the final concentration of 50ng/µl.
G. 1 Kb ladder
Take 100 µl of 1 Kb ladder (1 µg/µl) and 200 µl of 10x loading
buffer and mix with 1700 µl of distilled water to get final
concentration of 50 ng/µl.
Preparation of DNA
1. Prepare 10 µl of each DNA sample and digest the sample with
restriction enzyme EcoRI.
2. Make a reaction mixture with the following: 6 µl of sterilized
water + 2 µl of enzyme
buffer + 1 µl of spermidine + 1 µl of Eco RI
(10 u/µl) enzyme / sample.
For example, if there are five samples, make a cocktail with 36 µl
of sterilized water, 12 µl of enzyme buffer, 6 µl of spermidine and
6 µl of enzyme.
3. Add 10 µl of reaction mix to each sample and incubate at 37°C for
4. Load the digested DNA of each sample along with undigested DNA in
a 0.9% agarose gel. Both digested and undigested DNA of each sample
should be loaded side by side.
5. It is always advisable to load a DNA of known concentration for
6. Decide the concentration of each DNA sample based on the
intensity of the florescence due to ethidium bromide staining.
Estimation of DNA concentration through gel electrophoresis may
utilize undigested unknown samples for comparison with undigested
concentration standards. However, undigested samples tend to display
equally intense fluorescent bands so that subtle differences in
their concentrations are difficult to discern on the gel.
Molecular weight standards such as lambda
HindIII can be used as
concentration markers for plasmids and probe inserts. The precise
weight distribution of the different molecular size fragments
present in a volume of such standard loaded on the gel can serve as
markers for comparing with unknowns.
Preparing the gel
1. Prepare the electrophoresis unit, clean the casting tray, running
2. Add the correct amount of powdered agarose to a measured quantity
of electrophoresis buffer.
3. Heat the slurry in microwave oven until the agarose is completely
4. Cool the solution to 60°C and add 5 µI of ethidium bromide for
every 100 ml of agarose slurry (from a stock of 10mg/ml) (Follow
step 14 if ethidium bromide is not added at this stage).
5. Setup the mould (casting tray) on an equal plane so that the agarose will be distributed uniformly through out the mould. Check
the level using a spirit level.
6. Seal both the ends of
the casting tray with sealing tape.
7. Pour the agarose gel solution into the mould slowly without the
formation of any air bubbles.
8. Allow the mould undisturbed for about 30-45 minutes until the agarose solidifies completely.
9. Prepare the electrophoresis buffer (1x TAE or TBE) and fill the
Remove the sealers carefully and place the mould in the gel tank
having the electrophoresis buffer.
11. Allow the gel with combs for some time in the buffer, so that
the removal of combs will be easy without making any hole in the
Loading the gel
11. Samples are mixed with loading buffer and are loaded in the
slots of the submerged gel. The maximum volume of the sample to be
loaded depends on the slot size. Usually sample volume of 20-30 µl
is loaded in each slot.
12. Load the size marker (HindIII
λ or 1 Kb ladder) along with the samples.
Running the gel
1. Agarose gels for most purposes are run at room temperature. Run
the gel very slowly (20-30V/cm), if it is to be blotted. It is
advisable to run the gel overnight if the blotting is to be done.
Staining the gel
1. Stain the gel with
Ethidium bromide (1 µg/ml) solution
1. Resolve the gel under UV light and take the picture using the
Polaroid films (Type 57 or 667). The DNA will light up under UV
since it has the ethidium bromide.