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Transformation of the Host Cells
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Materials Required

 

 

  •   LB broth
  •   LA- Amp  plate
  •   Micropipette and tips
  •   Incubator
  •   Water bath
  •   Microfuge tube
  •   Calcium chloride treated competent cells
  •   L-rod

 

 

Reagents Required

 

 

  •  X Gal:   Make a 2% (w/v)  stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. The X-gal tube should  wrap with  aluminium foil in order to prevent the damage caused by light and store at -20°C.

 

  • IPTG:   Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG with  8 ml of distilled H2O. Prepare the aliquots and store them at -20°C.

 

 

Procedure

 

 

  1. To transform the CaCl2- treated cells directly, transfer 200 µl of each suspension of competent cells to a sterile, chilled polypropylene   tube  using a chilled micropipette tip.
  2. Add DNA (<50ng in a volume of 10 µl) to each tube. Mix the contents of the tubes by  gentle swirling. Keep the tubes in ice for 30 minutes. 
  3. Transfer the tubes to rack placed in a preheated 42 ºC circulating water bath. Keep the tubes in  rack for 90 seconds.
  4. Transfer the tubes to an ice bath immediately. Allow the cells to chill for 1-2 minutes.
  5. Add 800 µl of LB medium to each tube. Incubate the cultures for 45 minutes in a water bath at 37 ºC .
  6. After incubation, add 40 µl of X –Gal and 7 µl of IPTG to the LA-Amp plate.
  7. Add appropriate volume  of transformed competent cells into the plate.
  8. Spread  all the contents uniformly using an L-rod. Keep the plates at room temperature until the liquid has been absorbed.
  9. Invert the plates and keep for incubation at 37 ºC. Transformed colonies will  appear in 12-16 hours of incubation.          

 

 

Differences Encountered in Real Laboratory

 

 

1. After taking the competent cells from the freezer, it should thaw at room temperature.

2. There should not be much time lag between adding and spreading of EDTA ,Beta-gal and Transformed cells. If so, the components will not spread uniformly in the plate.

3. Make sure that the samples are uniformly spread in to the plate(should care the spreading).

4. Make sure that the plates with transformed cells should be in inverted position while incubation.

 

 

Help:

 

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