As one attempts their daily Sisyphean challenge of uncovering a new fact there is one truism, or platitude, that informs all of our efforts at troubleshooting an unanticipated experimental outcome. This can be paraphrased as:
The number one cause is pilot error.
Accumulated experience of bench scientists everywhere shows that their own human errors are the simplest explanation whenever they try to deduce what could have gone wrong. In the real world this translates into an operating axiom:
Don't waste time and energy coming up with new physical laws, or multi-step explanations that require you to remake and test all of your reagents, just repeat the experiment and see if this phenomenon is reproducible.
With that new information, that it is, or is not reproducible, you can decide if there is any hypothesis to test. But please, don't just take my word for it. Survey experimentalists, and the more experience they have the better.