Vectors
Virtual cloning
Simulate a restriction digest and ligation, size the construct, carry features across, and check the fusion stays in frame with the vector tags.
Reading frame check
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Map of the construct
The simulation assumes complete digestion and clean ligation. Methylation sensitivity, vector dephosphorylation and ligation efficiency are not modelled. Features falling across a cut site are not carried into the construct, since their coordinates no longer mean anything.
Formula
construct = vector backbone + insert fragment
- backbone
- the part of the vector outside the two cuts, which is kept
- fragment removed
- the part of the vector between the two cuts, which is discarded
- insert fragment
- the part of the insert between its own two cuts
- overhang
- the single-stranded end an enzyme leaves; ends must be complementary to ligate
Worked example
Directional cloning of a 300 bp fragment with EcoRI and HindIII
- circular vector of 512 bp, EcoRI cutting at 201 and HindIII at 267
- insert with EcoRI cutting at 11 and HindIII at 317
- The fragment removed from the vector is the span between the cuts: 267 minus 201, so 66 bp.
- The backbone that remains: 512 minus 66, so 446 bp.
- The insert fragment: 317 minus 11, so 306 bp.
- The construct: 446 plus 306.
Answer a circular construct of 752 bp, with both restriction sites recreated at the junctions
Common mistakes
- Choosing two enzymes with the same overhang for directional cloning. BamHI and BglII both leave GATC, so the insert goes in either way round and the vector can self-ligate. The tool warns when this happens.
- Forgetting that a restriction site at the end of a primer needs extra bases. Most enzymes will not cut a site flush with the end of a fragment, so three to six bases of padding are needed.
- Overlooking that the restriction site itself is translated in the construct. An EcoRI site is six bases, which is two extra codons in the fusion protein — and if the total is not a multiple of three, everything downstream shifts out of frame.
- Trusting the simulation instead of checking methylation. Dam and Dcm methylation in E. coli blocks enzymes such as XbaI and ClaI, and this tool does not model it.
Frequently asked
What exactly does the frame check tell me?
Three things: whether the 5-prime junction is in frame with the translation start, whether translation runs through the insert without hitting a stop codon, and whether the 3-prime junction stays in frame for a C-terminal tag. Those are the three failures usually discovered only after sequencing.
Can I clone with a single enzyme?
Yes. The vector needs one site so it is linearised, and the insert needs two so the fragment is released. Because both ends carry the same overhang, orientation is random and must be checked afterwards by diagnostic digest.
Why were some features not carried into the construct?
Features falling across a cut site are severed, and their coordinates would be meaningless in the new molecule, so they are deliberately dropped and counted.
How do I set the vector to insert ratio?
Take the sizes calculated on this page into the ng to pmol tool to get the masses needed for the molar ratio you want.