What exactly happens when competent cells like DH5ɑ are heatshocked with DNA present? How does the DNA get inside the cells?
Specifically, why are all the steps necessary? What if you heatshock right after adding DNA? What if you don't put on ice after heatshock? What does the calcium chloride do? What actually happens when cells are "competent"? What governs transformation efficiency (besides obvious things like amount of DNA or cells)?
To make it clear what I'm talking about, I use a protocol like the following:
- Take cells out of -80C and thaw on ice for 5 min.
- Add 1 ul (~500 ng) plasmid DNA to 50 ul cells, mix gently with pipette tip.
- Leave on ice for 30 min.
- Put in 42C water bath for 45 sec.
- Put on ice for 10 min.
- Add 950 ul LB, put in 37C for 1 hour.
- Spread 300 ul of the culture on LB-agar plates with appropriate selection.
I usually get thousands of colonies from this (in fact, often 1:10 or 1:100 dilution is necessary so I can actually get isolated colonies). Even if I skip the outgrowth, I still get hundreds.
I don't have my competent cell protocol, but basically I use that one rubidium chloride that everyone uses: DH5ɑ cells are washed with some buffers, suspended in a solution with CaCl2 and RbCl, then frozen in liquid nitrogen. I never actually measured, but usually the concentration of cells in the frozen aliquots is about 10 times as many as you would get from an overnight liquid culture (they are spun down and resuspended in a small volume).