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Blue middle tint in white colonies for Blue/White screening

XGal IPTG Blue/White

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#1 watsonkatherine

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Posted 23 July 2013 - 12:40 PM

I am using Blue/White screening to check if my plasmid tranformation into DH5alpha worked. As expected - I received all blue and all whtie colonies for one of my transformations. In my other transformation though, I got all blue colonies and white colonies with a tint of blue in the middle. I did store my plates ON 4C before picking colonies. The initial incubation period was for 16 hours at 37C. 100ul of 50ug/mL Xgal was used directily in the transformation products (not the LB agar + Ampicillin plates). 50ul of 20% IPTG was also added to the transformation products before plating.

Any idea why I am getting a blue color in the middle of ALL my white colonies for one of the inserts? As stated - there are solid blue colonies on those plates as well.

#2 watsonkatherine

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Posted 23 July 2013 - 02:41 PM

BTW - the insert where I received solid white and blue colonies was 1kb. The insert that I received solid blue and white colonies with a bluish tint in the middle is 400bp. Could the fact that this insert is a lot smaller be the issue as to why I'm not seeing solid white colonies?

#3 bob1

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Posted 23 July 2013 - 03:48 PM

Yes, that could be the problem - basically the insert is small enough that the ribosome is either reading through the stop codon or skipping the insert.




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