competent cells again!!

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Thu Jun 4 01:50:33 EST 1998


gwallmanning at my-dejanews.com wrote:
> 
> In article <l03130300b19b60eeb7ca@[134.174.248.196]>,
>   jaugliera at RICS.BWH.HARVARD.EDU ("John Augliera") wrote:
> cold CaCl2 and placing at -70C.  It makes no mention of adding a
> > cryoprotectant such as glycerol.  Would anyone know if this is OK!! or
> > would the addition
> > 10% glycerol adversely affect the competency or viability of the cells.
> >
> > Thanks
> >
> > John Augliera
> > Technician
> > Brigham and Women's Hospital
> > Boston,MA 02115
> 
> Adding 10% glycerol does not appear to affect the competency of the cells,
> although I would think that it would enhance the viability.  You could split
> a batch and treat half with and half without glycerol, and test competency
> and viability.  In the past I have found that it is better to filter
> sterilize the glycerol solutions than autoclave them as the autoclaving

Look out! I used to filter sterilize solutions for working with bacteria until
I found out that some batches of filters bleed out detergents that can lyse
your bacteria. So, it is best to test your batch of filters by filtration of
(autoclaved) LB (or your preferred medium) and inoculating with bacteria, and
doing the parallel assay without filtration. If growth is the same in both
cultures, filtration is safe and preferred.

Frank



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