making competent E coli DH5a cells - (Jan/30/2009 )

Ok I have a protocol for making these competent cells but I don't know what happens! Can anyone explain me what happens when you make your cells competent?

minemin on Jan 30 2009, 03:55 AM said:

The overall idea is to create holes in the outer membrane to allow the penetration of DNA species into the bacteria, you can achieve this by Chemical transformation or by electroporation

pesji on Jan 30 2009, 05:11 AM said:

Yes I know the principle but not the exact reaction, e.g.: we use MgCl2 en CaCl2 but I don't know why. If there is anyone who can explain this for me?

This is a good question, in fact I also tried to search the answer but the only thing I could find is that mechanism of calcium chloride-induced competence is still not completely understood. People tried to substitute Cacl2 with other salts but it dint work. I read it somewhere, that the process is physicochemical, rather than physiological. People speculate about the process using the knowledge of chemistry and physics. The phosphate backbone of DNA is negatively-charged, so it is likely that the calcium ions promote binding between DNA and the outer surface of the cell. since we incubate it on ice,this incubation reduces the thermal motion of molecules and so further promotes the binding process. On heat shock, the cell membrane alters in a way that allows DNA uptake.

Still would like to know more about it

Does anyone know what's the better method: electroporation or heat-shock? I used to use heat-shock method in my old lab because we didn't have an electroporator and I had minimal problems with cloning. But in my new lab they use electroporation and i'm having serious problems with cloning. Just wanted to know what everyones way is.

Lemmiwinks

P.s. First post! Whoop!

lemmiwinks on Jan 31 2009, 07:24 AM said:

As far as the efficiency is concerned, the electroporation is much better. On the other hand, the percentage of cell death after eletroporation is also much higher than this in heat-shock method. I used heat-shock in the past and now I use electroporation and I think electroporation is more convenient: faster to prepare the competent cell, less colonies needed for transformation screening.

Quasimondo on Jan 30 2009, 07:06 PM said:

I agree. Electroporation is better than chemical transfection. About 10 times to 100 times better depending on preparation.