replica plates - (Mar/15/2006 )

I have a big problem with some replica plates.
Ia must obtain yeast colonies without the KANr...i plated them on YPD and replicate them on G418 but, in more than 30 plates, i have no positive colonies.

can you tell me a method to increase the efficiency?


tnx

What have you done prior to plating your culture on non-selective media and replicating those plates to selective media?

What is the nature of the kan resistance in the original strain (e.g. plasmid borne, integrated into the genome, etc.)?

How many cfu per plate are you achieving on your non-selective plates?

they grew O/N in non-selective media (30C, shaker) but i tried also repeating inocula for 3 days 2 times a day.


KANr derives from integration of a linear cassette into yeast's genome

I plated on YPD about 100 - 150 colonies per plate.



i read about UV irradiation to increase the efficency but i do not like this technique

There is nothing wrong with your technique, provided the integrated cassette can be lost by a cross-out event.

When we wish to make a deletion mutant, we will integrate a suicide vector containing flanking DNA into the chromosome, then passage the culture for about a week without selection, plate to non-selective plates (generally 30 - 40 plates at ~ 200 colonies per plate) and replica plate these to selective media. We'll test each colony that has lost resistance to determine whether it's the mutant cross out or a wild-type revertant.

It's a pretty "stone knives and bear skins" method, but it works (there is a dearth of genetic tools available for our bacteria, so we do it the old fashioned way). We'll generally get 2 or 3 cross-out colonies per experiment. I usually keep passaging the cultures while I wait for the plates to grow and for analysis of the now-sensitive colonies (if any).

However, we know our plasmid can cross out via homologous recombination. By what method are you hoping your cassette will be lost? Or are you just hoping for a spontaneous point mutation?

I agree, I wouldn't use something like UV irradiation, it's too non-specific, and you'll never know if a secondary mutation occurred in your kan-sensitive strain.

Maybe you can get a bit more targeted by using a variation of what I've outlined above? Ligate two pieces of DNA flanking the cassette into a suicide vector, move that in, and screen for loss of the plasmid?