Home › Cell Biology › Restriction Digest Analysis
Steps
  1. 1 Understand restriction digest principles 00:05
  2. 2 Plan expected digestion results 00:58
  3. 3 Analyze undigested control lane 03:21
  4. 4 Run restriction digestions on gel 04:34
  5. 5 Evaluate single digest results 05:42
  6. 6 Analyze double digest bands 06:37
  7. 7 Confirm plasmid identity via EcoRI digest 07:05
  8. 8 Conclude plasmid identity verification 07:32
Cell Biology Addgene

Restriction Digest Analysis

Protocol
Difficulty
intermediate

Steps

1
Understand restriction digest principles

Learn why restriction enzyme digestion is useful for analyzing plasmid DNA, including how it reveals purity, confirmation state, and transfection efficiency. Understand the differences between supercoiled, nicked, and linear DNA conformations and their migration patterns.

▶ 00:05
2
Plan expected digestion results

Draw out the predicted band patterns for all digestion conditions before running the experiment. Map the expected products for single digests with AgeI and XhoI, double digest with both enzymes, and EcoRI digest which has two cut sites.

▶ 00:58
3
Analyze undigested control lane

Examine Lane 2 containing uncut plasmid DNA to identify supercoiled and nicked conformations. Note that supercoiled DNA runs faster (appears smaller) and nicked DNA runs slower (appears larger) than the equivalent linear DNA on the ladder.

▶ 03:21
4
Run restriction digestions on gel

Prepare and run the agarose gel with all digestion lanes, allowing sufficient time for band separation without rushing the electrophoresis. Include the size marker, undigested control, and all single and double digest samples.

▶ 04:34
5
Evaluate single digest results

Compare Lanes 3 and 4 (AgeI and XhoI single digests) to expected results, confirming a single linear 7.5 KB band in each lane. Verify band sharpness indicates a clean DNA preparation with minimal degradation.

▶ 05:42
6
Analyze double digest bands

Examine Lane 5 for the AgeI and XhoI double digest to identify the 7.2 KB and 300 bp fragments. Confirm the 300 bp band is fainter than expected due to its smaller size being 24 times less intense in a 1:1 molar ratio.

▶ 06:37
7
Confirm plasmid identity via EcoRI digest

Analyze Lane 6 containing the EcoRI double digest to identify the expected 5.4 KB and 2.1 KB fragments. Verify complete digestion by confirming absence of residual supercoiled or nicked bands.

▶ 07:05
8
Conclude plasmid identity verification

Compare all experimental results against expected patterns to confirm the isolated plasmid matches the known plasmid identity. Note the clean gel pattern with no unexplained bands indicates successful verification of the plasmid.

▶ 07:32

🚨 Failure Case Library (14) + Submit your own case

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
Double digest band pattern does not match prediction
Picked colonies grow normally but the diagnostic double-digest yields bands of unexpected size on the gel.
💡 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
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
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
severe
Incomplete Restriction Enzyme Digestion
Partial digestion observed with both expected fragments and uncut substrate visible on gel; incomplete linearization of plasmid DNA.
💡 6 · ✓ 6
moderate
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
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
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
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
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
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
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