Transformation of E.coli Cells

Transformation in E. coli DH5- α by electroporation

For electroporation following protocol can be used.

1.      Electroporation cuvetts 2 mm gap place on ice.

2.      Vials of frozen electrocompetent cells of DH5-α allow to thaw on ice.

3.      2 μl of ligation mixtures pipette into Eppendorf tube containing the competent cells and mixe gently with the pipette tip.

4.      The conditions for electroporation are:

Choose mode T                                               2.5 KV

Set resistance R                                               R5 (129 ohm)

Chamber gap                                                   2 mm

Set charging voltage                                       2.45 KV

5.      The electrocompetent cells containing the ligation mixture then transfer to

      electroporation cuvette.

6.      Pulse the mixture and add 1 ml of liquid LB medium immediately, mix gently and transfer to a 1.5 ml Eppendorf tube and incubate at 37 ⁰C for 45 minutes with vigorous shaking.

7.      Spread 100 μl of transformed culture on solid LB medium having antibiotic.

8.      After the liquid absorbed completely, seal the plates with sealing film and keep at 37 ⁰C in incubation for over night.

9.      Pick the Colonies with sterile toothpicks and culture in 3 ml liquid L.B medium containing relevant antibiotic.

10.  Keep culture  tubes at 37 ⁰C in water bath for over night with vigorous shaking.

11.  Isolate plasmid  and checked on 1% agarose gel.