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blue white screening


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3 replies to this topic

#1 sara.r

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Posted 05 February 2010 - 01:58 AM

hi
I clone a gene into pGEM T easy vector and transform it into TOP10 strain after 16 h incubation at incubator I got a lot of white clones, so I prepared a back up plate from 20 white clones and put it at incubator for about 20h but all of them turned in blue this time, I just dont know ehat happened, any suggesstions?
(I put the original transformation plate at +4 and there are still alot of white clones)

#2 phage434

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Posted 05 February 2010 - 05:39 AM

Were there some blue colonies on your initial plate? Perhaps the X-Gal was missing or degraded on that plate.

I'm a fan of S-Gal (Sigma) for blue/white screening (not that I do a lot any more). The color change is much more reliable.

#3 fishdoc

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Posted 05 February 2010 - 06:52 AM

hi
I clone a gene into pGEM T easy vector and transform it into TOP10 strain after 16 h incubation at incubator I got a lot of white clones, so I prepared a back up plate from 20 white clones and put it at incubator for about 20h but all of them turned in blue this time, I just dont know ehat happened, any suggesstions?
(I put the original transformation plate at +4 and there are still alot of white clones)



I've found that, depending on the vector copy number, it can take awhile for the blue to develop. Even after 16 hours of incubation, sometimes the blue is only barely noticeable, even with higher copy number vectors (I frequently use pBluescript). Maybe the difference between the 16 and 20 hr cultures is just the time to develop the color. I would try transferring the ones that are still white after sitting in the fridge. I think they'll be white on the next plate, as well. If not, then it could be something with the concentration of X-gal or IPTG.

#4 sara.r

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Posted 05 February 2010 - 10:10 PM

hi
I clone a gene into pGEM T easy vector and transform it into TOP10 strain after 16 h incubation at incubator I got a lot of white clones, so I prepared a back up plate from 20 white clones and put it at incubator for about 20h but all of them turned in blue this time, I just dont know ehat happened, any suggesstions?
(I put the original transformation plate at +4 and there are still alot of white clones)



I've found that, depending on the vector copy number, it can take awhile for the blue to develop. Even after 16 hours of incubation, sometimes the blue is only barely noticeable, even with higher copy number vectors (I frequently use pBluescript). Maybe the difference between the 16 and 20 hr cultures is just the time to develop the color. I would try transferring the ones that are still white after sitting in the fridge. I think they'll be white on the next plate, as well. If not, then it could be something with the concentration of X-gal or IPTG.


actually I pick up some other white clones after incubation in the fridge and put them for 20 h at 37 and the resulting clones are light blue! what do you mean by xgal iptg concentration?




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