Bacterial Transformation

What its for:

Introduce recombinant plasmid DNA into E. coli so that each transformed cell amplifies your vector, and use antibiotic selection to recover only those clones bearing the plasmid. This yields clonal populations for minipreps, sequencing, and downstream expression studies

Steps

• Thaw competent cells (ice, 10 min)

  • Keep 50 µL chemically competent E. coli on ice to preserve membrane permeability. ​

• DNA uptake (ice, 20 min)

  • Add 1–5 µL plasmid DNA (100 pg–100 ng), flick gently to mix—no vortex—and incubate on ice for 20 min to let DNA associate with the membrane. ​

• Heat shock (42 °C, 30 s + ice, 5 min)

  • Transfer tube to a 42 °C water bath for exactly 30 s to create transient pores; immediately return to ice for 5 min to reseal membranes. ​

• Recovery/outgrowth (30 °C, 20 min)

  • Add 350 µL room‑temperature outgrowth medium, then incubate at 30 °C with gentle shaking (250 rpm) for 20 min to allow expression of the antibiotic‑resistance gene. ​

• Plating (30 °C, 24 h or 37 °C overnight)

  • Warm LB‑ampicillin plates to 30 °C. Mix cells by flicking, plate 50–100 µL per plate, and incubate to form discrete colonies. ​

Application:

After site‑directed mutagenesis of your PCBP1 construct (GXXG→GDDG), you transform E. coli to amplify the mutated plasmid. Antibiotic‑resistant colonies are picked, grown, and mini‑prepped for sequencing to confirm the mutation before mammalian transfection.

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