|
WARNING. TAKE CARE AS ETHIDIUM BROMIDE IS HARMFUL,
AND GLOVES SHOULD BE WORN AT ALL TIMES.
1. Prepare 60 ml of 2% agarose in 1 x TBE buffer (Polymerase Chain
Reaction).
2. Either heat in microwave for approximately 2 min on full power
or place in a beaker of boiling water until melted.
3. Allow to cool to approximately 45°C and add 1 µl of ethidium
bromide (stock = 5 mg/µl) per 10 ml, to give a final concentration
of 0.5 µg/ml. This can be increased to 1 µg/ml if no ethidium
bromide is added to the buffer (see below).
4. Pour gel, insert well former (comb), and allow to set on a
flat surface for about 15 minutes.
5. Pour buffer 1 x TBE (containing 0.5 µg/ml of ethidium bromide,
i.e., 1 µl of 5 mg/ml stock to every 10 ml of buffer) into the
electrophoresis tank and remove comb from gel.
6. Prepare samples in tubes, or a multiwell plate, as follows:
1 µl of loading buffer
5 µl of PCR product
7. Prepare molecular weight markers as:
0.5 µl of molecular weight markersVI
(Boehringer)
1 µl of loading buffer
4.5 µl of water
8. Load samples into the wells formed in the gel. Molecular weight
markers can be loaded in both the first and last lanes.
9. Electrophorese at 100 volts for 20 minutes (minimum) or at
10 volts overnight.
10. View and photograph the gel on an UV-transilluminator (PCR
Reaction Products). USE UV SAFETY SPECTACLES
6x Loading Buffer
|
Reagent
|
Final conc. (%)
|
Amount (µl)
|
|
Glycerol
|
30
|
300
|
|
10% bromophenol blue
|
0.25
|
25
|
|
10% xylene cyanol
|
0.25
|
25
|
|
Water
|
|
650
|
|
Total
|
|
1000
|
|
