[Chlamydomonas] colony lifts
(by wirsch from cellbio.emory.edu)
Tue Feb 27 14:03:04 EST 2007
Has anyone has tried doing colony lifts on Chlamy? (Like how we do colony lifts for bacterial colonies or phage lifts for phage screening?). I was looking for a quick higher throughput method for screening for RNAi transformants, other than to try westerns on whole cell lysates or axonemes. We have about 1000 transformants (co-transformed with either pHyg3 for hygromycin resistance, or pARG7.8 for arginine) that we need to screen for RNAi of our gene.
Our attempts at colony PCR are inconsistent--even getting consistent PCR with mating type primers seems to be a bit troublesome in our hands, so we typically use purified genomic DNA for consistent PCR. So, I got to thinking about doing some type of colony lift and I don't know if this has been tried with Chlamy at all. Just wondering.
I may opt to redesign my RNAi construct to put in the pHyg cassette for hygromycin resistance, so I can do direct selection. Nonetheless, I still got to thinking about whether or not anyone has even tried doing southerns on colony lifts from Chlamy plates.
If anyone has any thoughts, that would be great.
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