Cloning, Yeast complementation

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Cloning, Yeast complementation

Post by molecule » Thu Apr 09, 2009 12:03 am

On a project that I have been working I have created mutations in cdc21 to look at how the mutations would affect the phosphorylation. After creating mutations and cloning the mutants to a suitable vector I need to study the complementation of temperature sensitivity of the mutated cdc21s to regular cdc21 /MCM4 . I’m confused about the concept of complementation and how I should get about doing the complementation tests using the regular temperature sensitive cdc21 and the mutated cdc21.
And I how I should confirm this by using a diploid.

Further I have created my mutations but I'm not sure as to how I should pick a vector for expression. Since I'm not very familiar with this field and any help is greatly appreciated.

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Post by MrMistery » Sat Apr 11, 2009 12:39 pm

i think all you are doing in the complementation study is to verify that you have mutated the proper gene. when you mate the mutant cells you created with cells that have a known mutation in the cdc21 gene, then at the nonpermissive temperature you should still see the mutant phenotype, if you made a mutation in the proper gene (this is because the new organism still doesn't have any wild type copies of the gene).

Regarding your choice of vector, it really depends on what experiments you want to do further on
"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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