In classic CaCl2-mediated transformation of E. coli (conventional method), plasmid DNA interacts with competent cells under which environmental conditions before heat shock?

Difficulty: Easy

Correct Answer: Low temperature and Ca++

Explanation:


Introduction / Context:
Conventional chemical transformation uses divalent cations to render E. coli competent to take up plasmid DNA. Understanding the temperature and ion conditions during the binding stage explains why a subsequent heat shock is required.


Given Data / Assumptions:

  • Method: CaCl2 chemical competence (not electroporation).
  • Focus: the environment while DNA associates with the cell surface prior to heat shock.
  • Heat shock at ~42 °C occurs after this interaction step.


Concept / Approach:
At low temperature (ice), Ca2+ neutralizes negative charges on DNA and lipopolysaccharides, allowing plasmid DNA to adhere to the cell envelope. The system is then briefly heat-shocked to create a thermal imbalance that drives DNA uptake through transient pores.


Step-by-Step Solution:
1) Competency induction: cells are incubated in cold CaCl2 to stabilize membranes and screen charges.2) DNA addition: plasmid is mixed with cells while kept on ice, maximizing surface binding.3) Heat shock: a short high-temperature pulse promotes DNA entry; this comes after, not during, the binding step.4) Therefore, the interaction environment is low temperature with Ca++ present.


Verification / Alternative check:
Standard protocols (Mandel–Higa, Hanahan) specify cold CaCl2 (often with additional salts) for binding, followed by heat shock; Mg2+ alone is not the defining ion in the classic version.


Why Other Options Are Wrong:
High temperature and Ca++: heat is used later for shock, not during initial interaction.High temperature and Mg++ / Low temperature and Mg++: Mg2+ may be present in some recipes, but Ca2+ at low temperature is characteristic of the binding step.


Common Pitfalls:
Confusing the binding step with heat shock, or assuming Mg2+ is the key ion for the conventional method.


Final Answer:
Low temperature and Ca++

Discussion & Comments

No comments yet. Be the first to comment!
Join Discussion