Hands-on plasmid construction guide

critical

Confirmed colony later shows recombination or deletion

Initial screening looked correct, but on re-verification the insert is missing pieces or the vector has rearranged.

💡 4 · ✓ 4

critical

Restriction Enzyme Complete Failure to Cleave DNA

No cleavage observed in restriction digest reaction; DNA remains uncut on gel, resulting in few or no transformants in downstream cloning applications.

💡 5 · ✓ 5

severe

Partial Digest with Multiple Uncut Bands

Gel shows mixture of fully digested, partially digested, and uncut DNA. Some recognition sites cleaved while others remain intact in the same molecule.

💡 5 · ✓ 5

severe

Incomplete Restriction Enzyme Digestion

DNA substrate is not fully cleaved after restriction enzyme incubation. Uncut or partially cut DNA bands persist on agarose gel alongside expected digestion products.

💡 7 · ✓ 7

severe

Few or No Transformants After Cloning

Bacterial transformation yields few or no colonies after restriction digest-based cloning. Vector may not be linearized or insert ends incompatible.

💡 4 · ✓ 4

moderate

Star Activity Producing Extra Bands

Extra DNA bands appear on gel beyond expected digest fragments, indicating cleavage at non-canonical recognition sequences due to relaxed enzyme specificity.

💡 4 · ✓ 5

moderate

Star Activity Generating Extra Bands

Extra bands appear on gel beyond expected digestion products. Enzyme cleaves at non-canonical sites with relaxed sequence specificity under suboptimal conditions.

💡 5 · ✓ 5

minor

Larger Than Expected Bands from Enzyme-DNA Binding

DNA bands migrate slower than expected molecular weight on gel, appearing larger due to restriction enzyme remaining bound to digested DNA fragments.

💡 2 · ✓ 3