Hello, I am doing a microsatellite PCR and the bands appear with a few definition, the electrophoresis conditions are the following, TBE buffer 0.5X pH 8.4 (according to Sambrook, Molecular Cloning), 45 mA constant or 100 V constant (I've try both conditions), 8% polyacrylamide gel (according to Sambrook, Molecular Cloning) (8 cm long), loading of pPCR 3uL + 1uL Gel Red [1: 2000]. What can be my mistake that the bands are thick and have little definition?
Thank you.
Make sure that the gel is cast with freshly made reagents especially the APS. Wash out the wells thoroughly with buffer before loading the sample. Use less dna ( smaller volume and lower amount of dna) in the sample and make sure that the sample and loading dye are well mixed before loading
When you see some pointy migration in the band pattern it's usually a sign of a dirty well. As Paul advice cleaning thoroughly the wells before loading you can use a syringe and flush the wells to remove any small debris of acrylamide (maybe due to a dirty comb or insufficient polymerisation)
Looks like dirty equipment. And you might have pulled the combs too soon - that can also lead to "wobbly" bands instead of ones that are well-defined.
- Badges
- Science method