3 Agarose Gel electrophoresis

Previous  Next

 

 

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
Ethidium bromide
Hind III
λ 1 Kb ladder

Composition of reaction mixtures

A.  Agarose (May change according to the electrophoresis unit)

Gel size (cm)

Agarose (0.8%)

Agarose (1.0%)

1x Gel buffer

Sample volume

11 14

0.80 g

1.00 g

100 ml

20 l

11 20

1.20 g

1.50 g

150 ml

20 l

20 20

2.40 g

3.00 g

300 ml

25 l

20 25

2.80 g

3.50 g

350 ml

50 l (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 of ddH2O

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

Caution: 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 4c.

F.  Hind III λ

1.  Digest 200 l of λDNA (0.25 g/l) using 25 l of HindIII enzyme. Incubate the reaction at 37c overnight.

2.  Test the DNA for complete digestion. If the DNA is digested incubate the mixture at 65c for 10 minutes.

3.  Distribute the 250 l to five 1.5 ml tubes each to have 50 l and make up the volume of each 50 l to 200 l by adding 20 l of loading buffer and 130 l of distilled water to make the final concentration of 50ng/l.

G. 1 Kb ladder

Take 100 l of 1 Kb ladder (1 g/l) and 200 l of 10x loading buffer and mix with 1700 l of distilled water to get final concentration of 50 ng/l.

Preparation of DNA

1.  Prepare 10 l of each DNA sample and digest the sample with restriction enzyme EcoRI.

2.   Make a reaction mixture with the following: 6 l of sterilized water + 2 l of enzyme buffer + 1 l of spermidine + 1 l of Eco RI (10 u/l) enzyme / sample.

For example, if there are five samples, make a cocktail with 36 l of sterilized water, 12 l of enzyme buffer, 6 l of spermidine and 6 l of enzyme.

3.   Add 10 l of reaction mix to each sample and incubate at 37C for 2-3 hours.

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 comparison.

6.  Decide the concentration of each DNA sample based on the intensity of the florescence due to ethidium bromide staining.

Notes:

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 platform, combs

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 dissolved.

4.  Cool the solution to 60C and add 5 I 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 gel tank.

10. 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 gel.

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 l 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 g/ml) solution

Photography

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.