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E. coli transformation

Updated 10/9/2020 02:15 PM
Step-by-step
by Franka Butzbach (created by Philip Schulz)

Introduction

Amplification fo the yeast plasmids

Materials

  • liquid LB
    • Agar
      • Antibiotics (GENT, SPEC and PARA)
        • Water (sterilized)

          Procedure

          Transformation

          1. take 30-50 microlitres of e.coli competent cells from the ultra freezer (-80*C). Store the tubes on ice for 5 minutes
          1. Add 1 microlitre of the plasmid, flick the tube to dsolve it and keep on ice for 2 minutes
          1. heat shock at 42*C in waterbath for 1 minute (DON'T exceed this time). Keep on ice after for 1 minute
          1. recovery: add 500-1000 microlitres of liquid LB (growth media) - incubate at 37*C for 60 minutes

          Preparing the agar with AB: GENT (12), SPEC (13) and PARA (7). Attention during agar microwaving (reduce microwave power)

          1. plating

          spin the tubes 30-60s at 6k rpm

          pour out supernatant LB

          resuspend in 50 μl in the BLB (liquid broth)

          plate together with the agar, 8-10 beads and the colonies

          1. incubate at 37*C (labelling with date & "iGEM")

          Comments

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