Possibly sample 9 has an RFLP base change creatibg the new large band compared to sam[le 8. The 2 bands created from the smaller band in sample 9 could be caused either by another rflp creating a new EcorV cut site or possibly just by a SNP in the bottom band that has formed a heteroduplex with the normal sequence so has a mismatch base causing the misnatched band to migrate slower so look larger than thenormal band. This option is more likely to show if you heat inactivated the enzyme after cutting

This due to digestion with EcoRV is likely due to variations in the DNA sequences present in each well, specifically the number of EcoRV recognition sites.

Lane 9 has probably more cuts due to more number of restriction sites. Another reason can be some contamination in the well or just simply some site-specific mutation in the pure sample.

Also that's not DNA ladder i hope.

Given that you stated all reaction conditions were identical (same enzyme, buffer, temperature, duration), the most probable explanations for the difference in digestion patterns between well 8 and well 9 must lie with the DNA samples themselves.

Let's analyze what the gel shows and propose explanations:

• Well 8 (Fewer cuts, larger bands): This pattern strongly suggests incomplete digestion of the DNA. This means the EcoRV enzyme did not cut all available recognition sites in the DNA.
• Well 9 (More DNA bands): This suggests a more extensive digestion, resulting in more fragments.

Here are the most likely reasons why well 9 would show more cuts than well 8, if both were processed in the same way, focusing on the DNA:

Why it's probably NOT a difference in enzyme or conditions (given your statement):

• You explicitly stated "same EcoRV enzyme, buffer, 37°C for 2 hours." This means that problems like enzyme degradation, incorrect buffer, wrong temperature, or insufficient incubation time are less likely to explain the specific difference between well 8 and well 9 if all samples shared the same master mix/incubation. If all samples used the exact same master mix, then enzyme activity should be consistent across wells.

In summary, the most plausible explanation, if your reaction conditions were truly identical, is that the DNA substrate loaded into well 9 is either a different DNA molecule with more EcoRV sites, or the DNA in well 8 was somehow inhibited, leading to incomplete digestion. You should carefully check the source and purity of the DNA samples used for wells 8 and 9.