Rubidium Chloride to make compotent cells

[Monique]

Does anyone know the molecular detail of how it works?
http://micro.nwfsc.noaa.gov/protocols/rbcl.html

It is a method to make E. coli cells competent so that they are willing to take up DNA from their environment. So I guess the RbCl disrupts the plasma membrane in a certain way.. but why the other specific salts? The glycerol must be make the cells freezable.. MOPS is a detergent..

I don't know exactly :P

[iansmith]

Rubidium chloride acts the same way as the clacium chloride. It is just an exotic reagent that supposebly increase the competence of the cell.

This is how it workes
The bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions. As the chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cells to swell and is necessary for the uptake of DNA. The exact mechanism of this uptake is unknown.

So Rubidium must have an impact on the permeability

His protocol is pretty fancy. In our lab we just use calcium chloride and then we add the glycerol as a cryoprotection

[Monique]

How does the CaCl protocol go? Does it require only a CaCl solution w/ glycerol?

I guess the RbCl is the foolsproof protocol [;)] I made the cells in an incredible rush on wednesday, transformed them on thursday and today I actually had colonies (which actually shouldn't have been colonies: digested vector ligated w/o insert).. or I must've contaminated them with an Amp resistant strain or something :P

[iansmith]

That is our protocol

Large scale production of competent E. coli DH5a

Competent cells were prepared by inoculating 200 ml of TY (0.8% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) with 4 ml of an overnight culture of E. coli DH5a. The culture was incubated at 37°C on a shaker at 200 rpm. The culture was grown to an optical density of 0.5 at 660 nm (Gilford Stasar II spectrophotometer Gilford Instrument Laboratories Inc., Oberlin, OH, USA). A 100 ml volume of the culture was then transferred to a 250 ml centrifuge tube and the culture was centrifuged (10,000 ´ g, 2 min, 4°C). The pellet was resuspended with 50 ml of cold (4°C) 50 mM CaCl2. The resuspended cells were put on ice for 10 min and then centrifuged again (10,000 ´ g, 2 min, 4°C). The pellet was resuspended with 30 ml of cold (4°C) 50 mM CaCl2 containing 15% (w/v) glycerol. The suspension was left on ice for 1.75 h and dispensed as small aliquots (~ 600 ml) for storage at -80°C.

[Moonbear]

Wow, sounds like that protocol just hits the cells with everything imaginable. I just buy cells that are already competent...since I don't work with those very often, it's easier...also handy when you're in a rush. I'm convinced most of molecular biology is just voodoo anyway [6)] Sure feels that way some days anyway.