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Transformation of E.coli

Updated 9/27/2021 07:56 PM
Step-by-step
by igem_lund

Introduction

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Materials

Procedure

  1. Take out plates with erythromycin and let them stand at room temperature for some time before using them. Also prepare a heating block at 42°C.
  1. Thaw the competent cells on ice for ~30 minutes
  1. Add 10-50ng of DNA (plasmid or ligation mixture) in a volume < 10µL and mix gently bur thoroughly. Do not vortex.
  1. Incubate on ice for 40 minutes (up to 2 hours is OK)
  1. Heat shock the cells at 42°C for 60 seconds.
  1. Place the cells on ice for a couple of minutes
  1. Add 400µL sterile LB medium
  1. Incubate the cells at 37°C for 45 minutes with shake.
  1. Spread out 50µL of the cells on LB plates with erythromycin

- If necessary: centrifuge and resuspend to dilute or concentrate, then plate.

  1. Leave the plates at room temperature until all liquid is absorbed
  1. Incubate plates at 37°C overnight

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