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UV Mutagenesis

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UV Mutagenesis

Postby sterl0601 » Fri Oct 28, 2005 3:36 pm

To all:
I am currently in the midst of research on Yeast proteins. I am trying to mutate them using UV. I have not had any success as yet. It seems that they are "burned up" in the protocol that I am using. Does anyone have any other UV mutagenesis protocol that I could use to create "successful" mutants? Any help would be greatly appreciated !

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Postby MrMistery » Fri Oct 28, 2005 7:17 pm

Doesn't the UV kill them? I would suggest using ionising radiation..
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
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Postby sterl0601 » Sat Oct 29, 2005 3:35 pm

I do want some of them to die. I am testing for TS mutants, to test the hypothesis that the translocon actively directs topology of membrane proteins or the conformation of secreted proteins. I am developing a library of temperature sensitive mutants in a haploid yeast strain harboring its only copy of sec61 on a plasmid which can be shuffled with a mutant library of plasmid borne sec61. I want to screen a large library of ts mutants en mass transformed with the library of mutant sec61 sequences to identify sec61-based suppressors. These suppressors, in the simplest scenario, function to facilitate folding of the unknown ts. If suppressors can be identified and their corresponding ts target genes isolated through a separate plasmid complementation screen, valuable insights might be gleaned about the language of topology and the machine that reads that language.
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