Team:Aachen/Wetlab/Protocol4

Transformation in E. coli

work space:

clean bench

conservation:

4 ℃ (after 16-18 hours at 37 ℃)

procedure

  1. Thaw 50 uL of E. coli Omp8 / 100 uL E. coli DH5α or E. coli BL21 (DE3) Gold on ice

  2. Add DNA (2-400 ng/uL: 50 ng if Plasmid DNA; 2 uL (~20ng) ligation mixture; 1-4 uL PLICing-reaction) to the competent cells and swirl gently

  3. Incubate for 15-30 min on ice.

  4. Perform heatshock of the competent cells using a preheated water bath at 42 ℃ for 90 s (Omp 8) or 45s (DH5-alpha, BL21).

  5. Samples were immediately cooled down 2 min on ice.

  6. Fill up the transformation mix to 1 mL with LB media or SOC media and incubate at 37 ℃ at 250rpm for 45 min (45-60 min) for recovery of the cells.

  7. Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench. (200 ul)

  8. resuspended pellet (from centrifugation of the leftover)

  9. Incubate the agar plates at 37 ℃ overnight (16-18 hours).

  10. Count single colonies of each agar plate on the next day.