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5-FOA selection


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6 replies to this topic

#1 wklee

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Posted 16 February 2002 - 05:16 PM

I need some help from anyone have any idea to solve my problem. I have some yeast strain (S. cerevisiae) need 5-FOA selection. After "URA3" popping out step I have some of colonies from SDC-5FOA plate. But these cells can grow uracil deficient plate too. is there any possibility cell can grow on both 5FOA containing plate and uracil deficient plate? Anyone have any idea, please let me know. That would help my work a lot.
thank you



#2 yuer

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Posted 20 March 2009 - 05:37 PM

Dear Everyone,

This is a very old post.This is really useful forum. I found it by searching 5 FOA. I realized that I am having the same problem. My yeast stain can grow on -ura plates and can also grow on 5 FOA plates. Strangely, after 5FOA selection, cells can grow on - ura plates again. Does anybody know why this could happen? I don't know if my 5 FOA plates didn't work. I dissolved 5FOA in DMSO. After autoclaving YPD, I added steriled 5FOA in YPD medium after cooling down to 65 degrees. Is this method OK? From some protocols, I knew that they used YNB + AA+ Glucose and 5FOA. I don't know if I can just YPD with 5FOA. Any comments will be greatly appreciated!

Have a great weekend!

cy

#3 perneseblue

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Posted 20 March 2009 - 06:24 PM

possible causes for this observation
gene silencing
mixed colonies

should also be noted that yeast cells can survive on FOA plates but not grow. So you have to be a bit careful with your streaking.

As for 5FOA in YPD medium, i don't think it would be a good idea. The uracil (from the yeast extract, which would be substantial) would be in competition with FOA when reacting with orotidine 5-phosphate decarboxylase (ura3 gene product). I have a feeling that selection will not be as good.
May your PCR products be long, your protocols short and your boss on holiday

#4 yuer

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Posted 23 March 2009 - 06:30 PM

possible causes for this observation
gene silencing
mixed colonies

should also be noted that yeast cells can survive on FOA plates but not grow. So you have to be a bit careful with your streaking.

As for 5FOA in YPD medium, i don't think it would be a good idea. The uracil (from the yeast extract, which would be substantial) would be in competition with FOA when reacting with orotidine 5-phosphate decarboxylase (ura3 gene product). I have a feeling that selection will not be as good.


Dear perneseblue,

Thank you so much for your reply for this topic and the topic i started last time. It is very nice of you. I deeply appreciate your help.
In the passed weekend, I was out of town. So I didn't use internet and read posts.

The information you gave me is extremely useful. Nobody in our lab worked on yeast before. Fortunately, there are so many experts including you in this bioforum.
If the cause for growing in both ura minus and 5FOA plates is gene silencing, do you know what i should do to get the URA3 mutant?

Thanks again,

Cy

#5 perneseblue

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Posted 24 March 2009 - 03:36 AM

do you know what i should do to get the URA3 mutant?


Well I would streak 100-200 FOA resistant colonies on ura- plates (with the same colony streak replicate on YPD) using sterile toothpicks. You should be able to isolate a real ura- colony this way.

A lower colony density on the FOA plate also helps. I noticed that FOA selection isn't so good once there colonies are too crowded.
May your PCR products be long, your protocols short and your boss on holiday

#6 yuer

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Posted 24 March 2009 - 05:54 PM

do you know what i should do to get the URA3 mutant?


Well I would streak 100-200 FOA resistant colonies on ura- plates (with the same colony streak replicate on YPD) using sterile toothpicks. You should be able to isolate a real ura- colony this way.

A lower colony density on the FOA plate also helps. I noticed that FOA selection isn't so good once there colonies are too crowded.


Hi, perneseblue,

Thanks a million for your very usefull tips and suggestions. You are my saver. I am trying the methods you told me. Hopefully, i will be able to isolate ura- strains. Thanks again. After reading the posts you gave in this bioforum, I deeply admire that you have such broad knowledge.

Cy

#7 molecule

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Posted 16 August 2010 - 02:11 PM

Hi,
I have a question on FOA selection. I have transformed a plasmid that contains a URA4 marker to yeast and I;m selecting for the intergration by plating on -URA plates. Eventually I need to loop out the URA4 containing piece and recover the plasmid intergrated Yeast strain. I'm not sure as to how to do the FOA selection to carry out the lopping out of the URA4. Can some one please advise me on this.




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