In blue white screening of recombinant and non recombinants using IPTG-Xgal I got colour series of blue colonies. Some are very dark and some are pale in color intensity. And I can agree for the colour difference at the edges. But in middle area of the plate also contain different colour colonies. Can anybody explain the reason?
Just don't care too much. Choose the white ones and be happy.
Reasons could be the uneven distribution of the substrates. Some colonies may have formed earlier and have produced more enzxme. If colonies are very dense you may have some mixed colonies or limited substrate. Light blue can also be the result of an in-frame insert.
Follow the other link also
https://www.researchgate.net/post/Any_advice_on_X-gal_blue_white_screening?_tpcectx=qa_overview_following&_trid=koAYcnDXgUxMWnBTuFdBLshM_
Thank you all.
Mr. Rao. I go through your link. And find if the insert Is low than 500 bp it may appear as blue colour.
Plese help me to figure out this. My insert size is 210 bp. Digestion of plasmid from pale white and white colonies couldn't obtain insert separate. Could see only linearized vector. I did digestion only for few colonies. Not all. What shall I do now? I didn't do colony PCR still.
do not worry about blue colour colonies. Just select white ones and go ahead.
just stick to the white colonies
the bluish colonies may as a result of two colonies overlapping each other or may as a result of uneven distribution of x-gal
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