Hi Lia, Would you provide more information about your transformation steps. If your incubation in 42C (heat shock is more than 1 min) the cells will die. Normally, 30 - 45 sec is preferred with 2 min of recover on ice before adding media for expression of antibiotic.

There's only one way to be sure and that's by performing some controls. Can you perform the same steps with empty vector and get colonies? Does it work if you use a control plasmid like pUC19? Do the plates you're using work with other cultures?

How do you recognize that the cells are allready dead before plating? What is the indicator?

Is your plasmid a high-copy number plasmid or a low copy number Plasmid?  Are you using the correct antibiotic at the correct concentration for your copy number?  Do a mock control (i.e. use 5 microliters of water instead of 5 microliters of DNA) in your transformations.  After 1 hr in the shaker make serial 1:10 dilutions of your mock and plasmid transformed E. coli cells.  Plate 100 ul of each on LB agar plates with and without antibiotics in them.  The mock control will give you an idea of the viability of your competent cells; they may be old.  The dilutions of both mock and DNA-transformed cells should give the same number of colonies on the LB agar plates without antibiotics.

were your cells stored for longer than 6 months?

If so they might have lost competence and you need to estimulate them again to become competent for transformation.

sorry I have no more ideas than those already mentioned.

best of luck!

If you see clearing/lysis in your cell suspension before plating, it's also possible that you acquired phage contamination somewhere along the way. 

Nothing to add to J. Harry Caufield (see above).

Dear all,

Thank you so much for your answers,

In my previous experients:

-I didn't include positive control

-the competent cells were stored more than 1 year old at -80°

-I incubated at 37°C

-before plating I noticed some precipitation ( therefore I thought cell death) and after plating it on agar for overnight at 37° I didnt' get any colonies.

I did again serials transformations using new competent cells and I included also the positive control with pUC plasmid and I incubated my plates at 30°C in stead of 37°C for 24 hours.

Surprisingly, I get now a lot of colonies.

I confirmed again that my cells didn't grow at 37°C, but I still don't know why.

So, thank you again for all your suggestions.