False-positives: restreaked yeast knockouts don't grow - (Oct/04/2009 )

I have been trying to make a yeast knockout using recombination of the kanmx6 cassette (i.e. PCR plasmid pFA6a-kanMX6, transform PCR product).

I have gotten colonies (~100/plate) growing on selection and the negative controls are clean. When I re-streak on selection, they fail to grow.

I'm wondering, could I be using too much plasmid template, which is being transformed into yeast, but since it's a bacterial origin they only grow on the first plate and not after restreaking?

I use the Qiagen PCR purification kits - do they filter out plasmid DNA?

Thanks

alexng on Oct 4 2009, 12:12 AM said:

How big are these colonies? Are they proper size colonies or micro-colonies? What kind of yeast are you using? Are you plating the same density of cells on the control and experimental plate?

In S. pombe requirements for an ARS signal is a AT rich sequence (>70%) and about >500bp. So it is rather flexible. If you are integrating DNA sequences, best to linearise the plasmid first before using it.That would usually solve problems of plasmids hanging around.

And yes, if the plasmid is smaller than 10kb, you can use a Qiagen PCR purification kit to pull down plasmid DNA. You are only using the silicon column within the kit to pull down the DNA. However you still need to make up the solutions from a alkaline lysis to isolate the plasmid DNA from the genomic DNA.