Biotechnology and Bioinformatics (1+1)

Resolution of restriction fragments on agarose gel

Materials

Electrophoresis buffer -50 X TAE

EtBr solution (10 mg/ml)

Agarose, electrophoresis-grade

6X loading buffer

DNA m.w markers

Method

1. Prepare the 1% gel, using electrophoresis buffer (final conc 1X) and electrophoresis-grade agarose by melting in a microwave oven, cooling to 55⁰C, add EtBr to a final conc of 0.5 m g/ml, pouring into a sealed gel casting platform, and inserting the gel comb.

2. After the gel has hardened, remove the seal from the gel casting platform and withdraw the gel comb. Place into an electrophoresis tank containing sufficient electrophoresis buffer (1X) to cover the gel ~1mm.

3. Take OD of the DNA samples at 260nm.

4. Prepare DNA samples (15-20 m g) with water and an appropriate amount of 6X loading buffer and load samples into wells with a pipette. Load appropriate DNA m.w. markers.

5. Attach the leads so that the DNA migrates to the anode or positive lead and electrophorese at 5 V/cm of gel.

6. Turn off the power supply when the bromophenol blue and xylene cyanol dye from the loading buffer have migrated a distance judged sufficient for separation of the DNA fragments.

7. Photograph a stained gel directly on a UV transilluminator.

8. Put a transparency on the gel and under UV-illumination , mark the m.w markers with the help of a transparency marking pen. Keep the transparency for alignment with the X-ray film of Southern blot.