Home › Cell Biology › Cloning With Restriction Enzymes

Steps

1 Understand restriction enzyme DNA cleavage 00:03
2 Identify ligatable DNA ends after cleavage 00:27
3 Determine compatible enzyme-vector end pairing 00:48
4 Verify recognition sites absent in insert 00:57
5 Examine methylation sensitivity of enzyme 01:10
6 Access online tools for enzyme selection 01:25

Cell Biology New England Biolabs

Cloning With Restriction Enzymes

Protocol

Difficulty

intermediate

Steps

1

Understand restriction enzyme DNA cleavage

Restriction enzymes recognize specific DNA sequences and generate double-stranded breaks in the DNA duplex. Cleavage produces fragments with either sticky ends (5' or 3' overhangs) or blunt ends.

▶ 00:03

2

Identify ligatable DNA ends after cleavage

After restriction enzyme cleavage, the 5' phosphates and 3' hydroxyls are maintained on DNA ends, leaving them ready to be joined together via ligation. This property makes restriction enzymes valuable for recombinant DNA technologies.

▶ 00:27

3

Determine compatible enzyme-vector end pairing

Select restriction enzymes that will produce DNA ends compatible with your chosen vector. This ensures the insert fragments can be properly ligated into the cloning vector.

▶ 00:48

4

Verify recognition sites absent in insert

Confirm that your selected restriction enzyme recognition sites do not occur within the DNA fragment to be cloned. This prevents unwanted internal cuts that would fragment your insert.

▶ 00:57

5

Examine methylation sensitivity of enzyme

Check the methylation sensitivity of your selected enzyme to ensure that host methylation by dam and/or dcm methylases will not block cleavage. This prevents failed digestion due to bacterial methylation patterns.

▶ 01:10

6

Access online tools for enzyme selection

Use NEB's online interactive tools to aid in selecting the appropriate restriction enzyme and to determine optimal reaction parameters. Visit CLONEWITHNEB.com for a complete list of available products.

▶ 01:25

🚨 Failure Case Library (13) + Submit your own case

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

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

DNA Smearing on Agarose Gel After Digestion

Restriction digest product appears as a smear rather than discrete bands on agarose gel, indicating DNA degradation or enzyme-DNA complex formation.

💡 3 · ✓ 4

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

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

Incomplete Restriction Enzyme Digestion

Partial digestion observed with both expected fragments and uncut substrate visible on gel; incomplete linearization of plasmid DNA.

💡 6 · ✓ 6

DNA Smear on Agarose Gel

Digested DNA appears as a smear rather than discrete bands on agarose gel. No sharp bands are visible across a wide molecular weight range.

💡 4 · ✓ 4

Failure to Cut Near DNA Termini

Restriction enzyme fails to cleave recognition sites located within 6 bp of linear DNA ends, such as PCR product termini. Full-length uncut product observed.

💡 3 · ✓ 4

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

Inefficient Cleavage Due to Single Recognition Site

Incomplete digestion persists despite adequate enzyme units and incubation time; some restriction enzymes require two recognition sites on the same DNA molecule for efficient cleavage.

💡 2 · ✓ 4

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

Enzyme-DNA Complex Causing Larger Bands

Larger molecular weight bands than expected appear on gel. Restriction enzyme remains bound to cleaved DNA substrate, retarding migration.

💡 3 · ✓ 3

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

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