Mutagenesis Blues

[Yasha Hartberg]

In article <kfischer-2910951436300001 at duckter.glaxo.med.ualberta.ca>,
kfischer at gpu.srv.ualberta.ca (Karl Fischer) wrote:

 > In article <Yasha-2910951434350001 at mac2wild2.tamu.edu>,
 > Yasha at bioch.tamu.edu (Yasha Hartberg) wrote:
 > 
 > >In short, each of the individual steps seems to be working
 > > fine so why in the hell can't we get any plaques?!!!
 > 
 > If you transform your host with freshly isolated dsDNA of M13 do you see
 > any plaques? If not then while your cells have previously tested positive
 > for competency (I'm assuming for a plasmid transformation), they might not
 > be expressing the F pilus though the F'-factor is detectable. In the past
 > I've made up an overnight "F+ feeder" culture of JM103 grown in
 > M9+Mg+Ca/1% casamino acids/0.001% thiamine/3% glycerol; following
 > transformation of the original competent cells I add 200 ul of these
 > feeder cells and then add top-agar. Has never failed to raise plaques.

No, I don't.  To summarize my last transformation:

                                        Result
     Transform with:  plasmid           antibiotic resistence
                      *M13 RF*          plaques
                      extension mix     no plaques
                      water             no plaques

In addition, I ran a control to be certain my antibiotic plates were
good.  Finally, to each transformation I added 100 uL feeder cells as you
describe, although the feeder cells were grown up in rich medium (TYE)
rather than minimal.  Since the feeder cells were an aliquot of the cells
I made competent, I don't expect this difference to be significant.  We
used to get plaques using our protocol without adding feeder cells at
all.  I'm stumped!
 
> One final caveat - DON'T VORTEX ANYTHING!!! Vortexing creates enough
> shearing force to "rip" the F pilus off the cells.

I'm always quite gentle with my competent cells, gently mixing by swirling
with the pipette tip.  I didn't know the shearing forces were that great
from vortexing, though.
 
> Hope you're up and mutating soon ;-)

Thanks for the encouragement, Karl.  This is getting DISCOURAGING!

Yasha Hartberg
Texas A&M University
"Now ritual is the husk of faith and loyalty, the beginning of confusion."  
Lao Tsu