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-   -   Problem using the X-alpha-Gal Protocol-at-a-glance (http://www.molecularstation.com/forum/yeast-forum/3666-problem-using-x-alpha-gal-protocol-glance.html)

larsinleeds 07-29-2008 03:17 AM

Problem using the X-alpha-Gal Protocol-at-a-glance
 
Hey all,

I am new in this forum hoping to find some answers.

I just started a Yeast 2 Hybrid Protocol using the Yeast strains Y187 and AH109 and the Plasmid pGBKT7(Gal4-BD) with my gene of interest.

One of the pre tests is the X-gal test. I have used the Protocol-at-a-Glance with alpha-X-Gal and both strains turned blue if they contained the Plasmid (empty strains were still white).

Now I am confused because I thought they have to be white as long there is a interaction between the Bait and the Prey Plasmid otherwise its auto activation. This would be worse because it means I couldn't do the Y2H-screening and I am running out of time.

I am also wondering why the colonies containing sequence A in the Bait Plasmid are much more blue than colonies with sequence B.

Does anyone has experiences with that? I just read that Promoter stuff again (Mel1 ...) but I am still lost.

Thank you all!

Lars

jennifer.chang 06-18-2009 08:05 PM

Re: Problem using the X-alpha-Gal Protocol-at-a-glance
 
I hope you were able to solve your problem or have it answered. My question to you is how blue are the strains with just the single plasmids in them? If it's not as blue as the p53 with large T antigen interaction, I would say you are safe. Also, I notice that after several days, all colonies turn a little blue. It's good to keep a continuous eye out on the plates to see if they are blue immediately (as soon as you see transformants) or if they turn blue eventually after 7 days.

I am having a sort of similar problem of the opposite sort. Except in my case, my cells don't turn blue enough (compared to the p53 & T interaction) so I am not quite sure if I am getting any sort of true interaction. I am also using the Clontech system. Could you PM me? I would really like to hear about your Y2H screen experiences as my Y2H just failed. I get no growth even on -L, -W, -H, X-alpha-gal, which is really surprising. I sometimes get a few colonies but again they are not very blue. They eventually do turn blue but never as blue as 53+T and they never have the ring of blue around them. I'm not willing to give up that my screen was completely negative so does anyone have any ideas on how I could troubleshoot?

ssherif08 02-05-2012 08:06 PM

Re: Problem using the X-alpha-Gal Protocol-at-a-glance
 
Guys, I don't have any problem getting blue colonies with p53-T7 interaction but I didn't get any interaction with my proteins. The blue colonies for p53-T7 appear at the second day of incubation at 30. Today is the third day and no colonies appear for my proteins. Only in one condition, I found only one colony and is a white one. My questions are:
- How long it may take to get positive colonies on selection medium containing eurobsidin
- What does it mean to get a white colony?
your suggestions will be appreciated a lot.
Sherif


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