Team:METU Turkey/Results Discussion/Agarose Gel Electrophoresis

From 2010.igem.org

MAIN STEPS/TIME TABLE
- 1% gel preparation [30 min]
- Running [1 h]
- Visualization [15 min]

MATERIALS
- Electrophoresis tank
- Power supply
- Transilluminator
- Appropriate comb
- P10 and P100
- DNA Ladder 100-10 kb (Fermentas #SM0331)
- Agarose
- Loading dye
- TAE Buffer (1X)
- Distilled water [M14]
- Sterile dH2O [M14]
- EtBr
- Parafilm

SOLUTIONS
TAE buffer
- Stock TAE electrophoresis buffer (50X)
- Use 1X TAE
- 20 mL TAE
- 980 mL dH2O
1% Electrophoresis gel
- 0.5 gr Agarose
- 50 mL 1X TAE buffer (1%)

CHECK-LIST PROCEDURE
- Mix 2 uL DNA + 3 uL sterile dH2O + 1 uL loading dye on the parafilm
- Load sample to the wells of 1% gel
- Adjust the voltage of power supply to 75 V
- Adjust the time of power supply to 65 min
- Check transilluminator
- After running of the samples record the gel image [go to SOPs-experimental for application of camera and record of image]

NOTES
- Before adding of EtBr make sure the temperature of gel solution, its temperature should not be too high.
- Make sure the voltage is at the correct settings at all times and always use enough buffering solution to cover the gel in order to prevent inconsistent readings.

TROUBLESHOOTING

Missing Bands
- Use a lower voltage or decrease the electrophoresis time if smaller bands are missing since this may indicate that they were pushed off of the gel. However, increase the electrophoresis time if larger bands are missing which means that the components have not separated yet.

Smear
- Check sample for nuclease contamination, buffering conditions and excess salt or protein. If you still see smeared results, decrease the amount of sample used.

Non-existent or Faint Bands
- Increase amount of sample or time of electrophoresis at a lower voltage.