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Ligation: restriction cloning

The shelf keeps its parts from one exercise to the next, so each exercise says what to remove first. Four short exercises on the Bench: the directional case that works, the mismatch that cannot, the single-enzyme case that needs dephosphorylation, and a real gene from NCBI.

Exercise 1 of 4: directional cloning with EcoRI + BamHI

  1. Open the Bench and press Clear shelf, then open pLig-Vector.gb and Insert-EcoRI-BamHI.gb.
  2. In the vector's tab open the Enzymes tab and click Hide all. (A file you open opens with every single cutter ticked, and the digest follows the ticks and nothing else.)
  3. Tick EcoRI and BamHI.
  4. Open the Cloning tab (picker: Digest).

    You see two fragments: 2,640 bp (BamHI 5′ GATC at its left end, EcoRI 5′ AATT at its right: the backbone) and 24 bp (EcoRI 5′ AATT to BamHI 5′ GATC: the stuffer, with the PstI site in it).

    If instead you see one fragment of 2,664 bp, only one of the two enzymes is cutting; check that both are ticked.

  5. Click Add on the 2,640 bp row.

    You see Added ✓ and the Bench count goes to 1.

  6. In the insert's tab open the Enzymes tab, click Hide all, and tick EcoRI and BamHI.

    You see 801, 11 and 7 bp. The 11 and 7 bp pieces are the padding with a blunt end; the 801 bp piece is EcoRI 5′ AATT to BamHI 5′ GATC and carries synthORF1.

  7. Add the 801 bp piece.
  8. Open the Bench tab and choose Ligation. Untick nothing; keep Circular product ticked.

    You see two parts on the shelf, the backbone first, then the insert.

  9. Check the junctions. Nothing needs flipping with ⇄.

    You see backbone to insert ✓ (EcoRI to EcoRI) and the closing junction ✓ (BamHI to BamHI).

  10. Read the Product line.

    You see 3,441 bp, circular, carrying the synthetic promoter, the synthetic terminator, AmpR, ori and synthORF1.

    Why

    MCS is there twice, as two partial pieces: the cut ran through it, and the 24 bp stuffer with the PstI site is gone.

  11. Click Assemble.
    • The new document is 3,441 bp (2,640 + 801) with synthORF1 on the forward strand, the same way as the promoter.

    If not, go back to the junction line: a ✕ there means the ends do not match.

  12. Open the new document's Enzymes tab.

    You see EcoRI and BamHI ticked, each cutting once. PstI cuts it not at all (it cut the vector only inside the stuffer); SacI cuts it twice (once in the backbone, once in the ORF).

Optional, a miniprep check: Check by digest is on the Bench, under the product's map, before you press Assemble (the assembled document has no such panel). A vector cut with two enzymes cannot close on itself, so the Bench says there is no empty vector to compare with and ranks the product's own lane, clearest first. With the bundled enzyme set the first rows are MluI: 2,876 + 565 bp, HpaI: 2,741 + 700 bp, ScaI: 2,566 + 875 bp, SacI: 2,541 + 900 bp. The vector alone with SacI is one band, 2,664 bp: that is how a miniprep digest of this clone differs from the empty vector.

Exercise 2 of 4: negative control, ends that do not match

  1. Close the Insert-EcoRI-BamHI tab. (The shelf is separate from the tabs, so this changes nothing there; it just keeps the tab bar tidy.) Open Insert-PstI-BamHI.gb.
  2. In its Enzymes tab click Hide all, then tick PstI and BamHI.
  3. Add the 797 bp piece (PstI 3′ TGCA to BamHI 5′ GATC).
  4. On the Bench, remove the earlier insert with its ✕.

    You see the 2,640 bp backbone first and this insert second on the shelf.

  5. Read the junctions.

    You see the junction from the backbone to the insert say ✕ ends do not match, the closing junction (BamHI to BamHI) ✓, no Product line, and Assemble off.

    Why

    The backbone ends in EcoRI (AATT, 5′ overhang), the insert begins with PstI (TGCA, 3′ overhang): those cannot pair.

  6. Press ⇄ on the insert.

    You see both junctions say ✕ (BamHI against EcoRI, PstI against BamHI).

    Why

    No orientation works, which is the point: incompatible ends are never rescued by flipping.

  7. The fix, as a designer would: put the vector's PstI end to use. Remove the backbone from the shelf with ✕.
  8. On the vector, untick EcoRI and tick PstI (BamHI stays ticked).

    You see fragments 2,656 and 8 bp.

  9. Add the 2,656 bp backbone, and put it first.
    • Both junctions are ✓ and the product is 3,453 bp, circular, with exactly one PstI site.

    If not, check that the backbone is first on the shelf and the insert second.

The vector's two ends and the insert's two ends must be the same pair: with EcoRI on one side and PstI on the other, the insert cannot go in at all.

Exercise 3 of 4: one enzyme, why dephosphorylate (−P)

A vector cut with a single enzyme has two identical ends and can close on itself, which is the usual empty-vector background. A phosphatase takes the 5′ phosphates off, so it cannot.

  1. On the Bench remove the Exercise 2 parts with ✕ (or press Clear shelf). Open Insert-EcoRI-EcoRI.gb (and the vector). On both, tick EcoRI only.

    You see for the vector one fragment, 2,664 bp, EcoRI 5′ AATT at both ends; for the insert the 801 bp piece, EcoRI at both ends.

  2. Add both, vector first.
  3. On the Bench choose Ligation, and untick the insert so only the vector is in the reaction.

    You see the closing junction ✓ and a product of 2,664 bp: the vector closed on itself, with no insert.

  4. Tick the insert again, and press −P on the vector's shelf row.
  5. Untick the insert once more.

    You see the vector alone say both sides dephosphorylated, and Assemble is off.

  6. Tick the insert again.

    You see the junctions ✓ ✓ (the insert still has its phosphates) and a product of 3,465 bp, circular.

    Why

    Only the vector lost its phosphates, so the insert can still ligate into it but the vector cannot ligate to itself.

  7. Press ⇄ on the insert.

    You see the same 3,465 bp with synthORF1 on the reverse strand (as shelved, it was on the forward strand).

    Why

    Orientation is not decided by the enzyme. On a bench you would screen for the right one.

Optional extra, a miniprep check digest: repeat the steps above without −P and, on the Bench, look at Check by digest under the 3,465 bp product, before Assemble. After Assemble the product opens with EcoRI ticked, two sites. The vector is the longest part, so it is the empty vector (cut once with EcoRI and closed on itself, 2,664 bp). The first rows: EcoRI: 2,664 + 801 bp | empty 2,664 bp and SacI: 2,565 + 900 bp | empty 2,664 bp. An insert-carrying colony shows the 801 bp band with EcoRI, an empty one only 2,664 bp, which is why −P matters.

Exercise 4 of 4: a gene from NCBI, human insulin (INS)

Accession NM_000207 (NM_000207.3, human insulin, mRNA, 465 bp; its CDS is 333 bp, the 110 residue preproinsulin plus the stop codon). It has no EcoRI or BamHI site in the CDS (checked), which is why EcoRI + BamHI will work.

  1. Open pLig-Vector.gb and leave it in front.
  2. Open the Primers tab (sidebar) and click Design primers….
  3. In the workspace, switch Mode to Clone an insert into this plasmid.

    You see Where on the vector: with nothing selected, it takes the feature named MCS.

  4. Under What to insert choose Gene from NCBI…, type NM_000207 in the box and press Add. Only that accession is sent to NCBI.

    You see the record open as a tab of its own, and its CDS becomes the insert: 333 bp, starting ATGGCCCTGTGGATGCGC and ending ...CTGGAGAACTACTGCAACTAG. The workspace stays on the vector.

  5. In Enzymes click the EcoRI · BamHI row. It is listed first, the other usable pairs after it. Leave Padding (6) and Junction at their defaults.
  6. Click Design primers.

    You see PCR 357 bp, one caution warning, and no red ones.

    The primers, written as ordered (tail in lower case):

    INS-F-EcoRI  5′ gttctggaattcATGGCCCTGTGGATGCGC 3′              (annealing part 18 nt, Tm 60.9 °C)
INS-R-BamHI  5′ ctaataggatccCTAGTTGCAGTAGTTCTCCAGCTG 3′         (annealing part 24 nt, Tm 57.7 °C)
    Why

    The caution says no annealing part at the insert's ends meets the settings; nearest is "GC content above 65%": the start of the insulin CDS is GC rich. The reverse primer's annealing part includes the stop codon (CTA is the reverse complement of TAG).

  7. Click Simulate. It runs PCR on the record, the EcoRI + BamHI digests and the ligation into the vector's backbone, and opens the product as a new document.
    • The product is 2,979 bp, circular.
  8. Open the product's Enzymes tab.

    You see EcoRI and BamHI ticked, the two enzymes the primers use, one site each. PstI does not cut this product at all.

    • One EcoRI site is just before the ATG and one BamHI site just after the stop codon.

    The product carries the vector's features and the record's annotation (INS, insulin preproprotein, sig_peptide, proinsulin, insulin B chain, C-peptide and the exons), plus the two primers as features, named after the record's feature (insulin_preproprotein-F-EcoRI); the primer names above are labels for this page.

Optional, a miniprep-style check: which single enzyme tells a colony that took the insert from one that did not. The enzyme with the clearest bands for this product is MluI, which cuts it twice: 2,414 + 565 bp (the same ranking function the Bench's Check by digest uses). That panel is not on screen here: this product came from the primer workspace's Simulate, not from a Bench reaction. Use the product's own Enzymes tab:

  1. Click Hide all, so EcoRI and BamHI stop counting in the digest.
  2. Cuts is a remembered setting. If it says Once, MluI is not listed, because it cuts twice. Change it to Twice, Once or twice, 3 times or fewer or Any number; Filter (the name box) narrows the list further, so type MluI there to find it quickly.
  3. Tick MluI. Fragments from ticked enzymes reads 2,414, 565 bp, two bands on the gel lane.

Done when:

Next: Golden Gate: one gene into one vector, or back to the tutorials index. Numbers off? See If a number does not match.

How it works

Background: Ligation, The shelf and Digest.