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transformation and counter selection of yeast mutants


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#1 yeastresearcher

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Posted 10 July 2011 - 03:58 AM

Hi

I have done yeast transformation since my plasmid has ura3 i have grew them on - ura plates I want to remove ura by homologous recombination. i then grew them on ypd media and plated on 5 FOA. should i select single colonies of 5- FOA and plate them again on 5 foa to get pure culture of recombinants that lack ura or what shoudl i do can anybody suggest me the protocol to get pure recombinants?

#2 pito

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Posted 10 July 2011 - 02:03 PM

Hi

I have done yeast transformation since my plasmid has ura3 i have grew them on - ura plates I want to remove ura by homologous recombination. i then grew them on ypd media and plated on 5 FOA. should i select single colonies of 5- FOA and plate them again on 5 foa to get pure culture of recombinants that lack ura or what shoudl i do can anybody suggest me the protocol to get pure recombinants?


Yeah, it would be best to plate single colonies out again. To rule out any yeasts cells that were able to suvrive because they landed on other colonies or whatever reason.
Its a double control + this way you can select single colonies better.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 yeastresearcher

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Posted 11 July 2011 - 06:46 AM


Hi

I have done yeast transformation since my plasmid has ura3 i have grew them on - ura plates I want to remove ura by homologous recombination. i then grew them on ypd media and plated on 5 FOA. should i select single colonies of 5- FOA and plate them again on 5 foa to get pure culture of recombinants that lack ura or what shoudl i do can anybody suggest me the protocol to get pure recombinants?


Yeah, it would be best to plate single colonies out again. To rule out any yeasts cells that were able to suvrive because they landed on other colonies or whatever reason.
Its a double control + this way you can select single colonies better.



Thank you ...




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