Plasmid Cloning: Restriction, Ligation & Transformation

1. Restriction Digest

Vector cut with

Insert cut with

GAATTC ↔ GAATTC — sticky ends match

2. Ligation

Insert : vector molar ratio 3.0 : 1

3. Heat-Shock Transformation

42 °C heat-shock duration 45 s

Ready — press Plate Transformation to spread bacteria on the ampicillin plate.

Live Readouts

Recombinant fraction

—

Transform. efficiency

—

Colonies on plate

— green / — white

How it works

Molecular cloning chains three independent physical steps; this sim models each with the formula real protocols are tuned around.

1. Digestion — a restriction enzyme cuts DNA only
   at its palindromic site, leaving a sticky end:
   EcoRI  G^AATTC     BamHI  G^GATCC     HindIII  A^AGCTT
   Vector and insert must be cut with the SAME enzyme
   (or compatible ends) or T4 ligase cannot join them.

2. Ligation — recombinant fraction rises with more
   insert relative to vector, saturating near ~90%:
   f_recomb(r) = 0.93 · r / (r + 1.8)      r = insert:vector ratio
   (incompatible ends collapse this to ~2% background
   from imperfect blunt re-ligation of the vector alone.)

3. Transformation — heat shock at 42 °C transiently
   opens pores in Ca2+-treated E. coli membranes; too
   short and pores don't open, too long and cells die —
   efficiency follows a bell curve peaked near 45 s:
   E(t) = E_max · exp( -(t - 45)^2 / (2 · 18^2) )   [cfu/µg]

Green colonies took up a recombinant plasmid (insert + vector) — the desired clone.
White colonies took up a self-ligated vector (no insert) — still ampicillin-resistant, but not what you wanted.
Ampicillin selection kills every cell that received no plasmid at all, so only these two outcomes ever appear on the plate.

Recombinant colony Self-ligated (no insert)

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