growing cometent cells - (Oct/11/2013 )

HI all,

during resistance plasmid growth in transformation step, protocols usually say shake growth for 1-2 hr at 37 deg C after heat shock. when i grow it without shaking, i get colonies, when i grow with shaking, very very few colonies. although neither times i had any insert, but rest that for now.  do you think i should grow stationary culture or shaking culture? and sometimes people say dh5alpha is very delicate and you need to keep on ice ass soon as its out of freezer and pipette gently to not kill the bacteria etc etc, which makes me think be ther reason why shake culture might be a problem but then they also say it is a very robust organism and so on. am confused! ! what is actually? and most importantly shake cuture or stationary?

thanks,

For maximal survival of the cells when thawing, thaw them as fast as possible in a 37 degree water bath or in your hand.  This prevents ice-crystal formation that can damage the cells if thawed slowly (i.e. on ice).

The growth following heat shock is to allow time for the expression of the antibiotic resistance genes, and as such it is important that this step NOT include antibiotics.  Shaking allows aeration of the culture, which is optimal for E. coli growth, however, for this short period, and given the small volume the cells are in, there probably won't be much difference between shaking and non-shaking.