Hi everyone, I’m trying to troubleshoot an issue I observed in my agarose gel image and would really appreciate your insights.

In the gel photo, the first lane is a DNA ladder, the second and third lanes are two different inserts, and the fourth lane is the vector. I took this picture before ligation to check the quantity of each DNA component.

In the fourth lane (the vector), I observed significant smearing. I'm wondering what might have caused this and how I can fix or prevent it.

Here are the details of the experiment:

  • The vector was extracted from E. coli DH5α using a mini-prep kit.
  • Double digestion was performed using BamHI-HF® and XbaI (both from NEB).
  • I used 1 µL of each enzyme, and approximately 10–15 µg of vector DNA.
  • The total reaction volume was 50 µL.
  • Digestion was carried out overnight.

Any suggestions or explanations would be greatly appreciated. Also, apologies for any awkward English—it's not my first language.

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