Transformation - (Nov/12/2005 )

Hallo!

I am trying to transform a ligation (13kbp) into E.coli. The colonies are growing on LB medium with Kanamycin but I can not isolate the plasmid, the control worked.
I used the E. coli strain TOP 10. I made them competend by centrifuge them at 4°C and resudpending the cells in 100mM ice-cold CaCl2. They were stored at -80°C. For the transformation I used a standard heat shock transformation protocol.
The vektor I used for ligation was pCambia 2301. I couldn´t make a colonie PCR to test success of ligation because the insert is an element of E.coli.

With which E.coli line have you made good experiences in transformation?

Hi, waidhouse! Can you give us a bit more information about your experiment?

What strain of E. coli are you using?

How did you make them competent?

Is this a competency transformation or an electroporation?

What vector are you using, and what selection media?

How did you perform your transformation?

Do your controls work?

Hi waidhouse, indeed you need to give us more information on your competent cells.
How competent are they?
Everytime I make competent cells I test the cell competency. Have you done that?

Hi again,

I checked in my lab book. Usually I get cells that are 1 or 1.5 million cfu/ug DNA/ 50ul cells

You can easily check the competency of the cells you made, if they are bellow the value I just mentioned, then you should check you protocol (check in Sambrook) and make new cells.
If they are competent enough, then you know you might have another problem.

To check the competency of my cells I transformed 50 ul cells with 0.1 ng pUC18 plasmid.

Which control are you using for the transformation, is it a plasmid as big as 13kb? Or is it much smaller? A ligation is always more difficult to transform than just pure plasmid.

Success.

I haven´t checked the compentency of my own cells but if have made a transformation with competent cells from intivrogen (also TOP 10) with no results.
The control plasmid had a size of 11,6kb.