Technique / Molecular Biology / Cloning library construction and screening
Blue / white colonies selection problem
Blue / white colonies selection problem
I am using blue/white selection to choose colonies with my insert.
Unfortunately, my white colonies don't have inserts. What could be causing this. ... ...
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Principle of blue white selection of positive recombinant colony: Plasmid contains bacterial lacZ gene can produce b-galactosidase (b-gal) if its ORF isn't interrupted by insert DNA. Together with alpha peptide expressed by hosting e.coli strain, functional b-galactosidase can be produced by transforming wild type lacZ containing plamid into e.coli cell and the b-galactosidase can turn the X-gal into a blue corlor products. When a foreign DNA is inserted into the multiple cloning site (MCS) within the lacZ gene, the open reading frame of lacZ is changed and will not produce b-galactosidase therefore the recombinant colony will be in white corlor. Figure illustration
Last update 17-Jul-2002, Rating of 19 votes.
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By Tuhin
on 20-Feb-2009
Principle of blue white selection of positive recombinant colony: Plasmid contains bacterial lacZ gene can produce b-galactosidase (b-gal) if its ORF isn't interrupted by insert DNA. Together with alpha peptide expressed by hosting e.coli strain, functional b-galactosidase can be produced by transforming wild type lacZ containing plamid into e.coli cell and the b-galactosidase can turn the X-gal into a blue corlor products. When a foreign DNA is inserted into the multiple cloning site (MCS) within the lacZ gene, the open reading frame of lacZ is changed and will not produce b-galactosidase therefore the recombinant colony will be in white corlor. |
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By Abhay
on 07-Nov-2008
Hi... |
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By Paul
on 23-Oct-2008
I used to do X-gal/IPTG screening in old times. I used to get all exited about getting only white colonies. However, it turned out to be taking some time to develope blue colors by colonies containing vector only. When I leave the plates in 4 degree (refrigerater) for a few hours, the colonies without transgene will turn blue (a little faster than in RT). |
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By Y2H
on 19-Apr-2008
Could you please help me with this confusing thing? I did pGEMTeasy cloning and got no blue colonies. It's too good to be true. Thus, I assume that my Xgal has some problem. I plated my labmate's blue colonies on my Xgal plates (the same I used for those cloning) and found all of blue colonies. Again, I set up cloning with only pGEMTeasy, buffer & T4 ligase without any inserts and once again I got no blue colonies. Because there are no inserts, there must be full of blue colonies or no colonies at all (if my transformation is bad) but what i've got is all white colonies. I've just bought the pGEMTeasy cloning kit few days ago from the DNA core next to my lab and the expiration date is Feb2011. Could anyone please give me any suggestions? The confusing results really drive me crazy. I already did 30 cloning with the same results- all white colonies. Thank you sooooooo much! |
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By Elhaseen Elesaid
on 06-Mar-2008
Hi |
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By RAJNI
on 31-Jan-2008
hi this is quite possible in some exceptional cases when the inserts size is very small,but the colony color could be light blue in that case |
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By ravendra
on 01-Jan-2008
i am doing homology modelling.i did not know how to generate loop building.can any one suggest me |
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By Annie
on 20-Nov-2007
Hi Guys, plz help. I am in the middle of the park. I just cloned 30 genes using pgemT-Easy vector. Now only two genes I have amplified from othe samples are not giving me the right answer as being negative in the white selection of colonies. I cheched my Pcr product, but looks, the band is so faint a bit. i do not know if really, thats why I am failing to get a recmbinant with the correct size or what. this are my last samples to clone. Plz help.Annie. |
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By Annie
on 20-Nov-2007
Hi. the reason is thta, blue colonies are originated from the X-gal solution if I am not wrong. leaving your plates in room temperature will still give your colonies a propagation to grow, that is why they turn to be blue. Aslo, growing bacteria for a vry long time will result in degradation of the palsmid DNA, that is why they are turning blue. maybe, after 12 h inc, plz store them in 4 ded c, to avoud discrepencies. also mark your white colies as possible. |
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By vijay
on 24-Oct-2007
how can maintain the plasmid in the alpha DH5 which has the t7 promoter mostly used for expression |
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By K.harsha Vardhan
on 02-Sep-2007
Dear sir, |
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By Kapil
on 04-Jul-2007
Dear Sir, |
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By sanjay
on 26-Mar-2007
Dear sir/madam.. |
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By Nguyen Hung Anh
on 06-Dec-2006
Can I select the X-gal screening for pGEX4T-3 in DH5alpha /BL21 strains. |
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By vrinda
on 01-Jun-2006
Iam doing tranformation of E.coli DH5 alpha with pUC18. I would like to know whether the transformed colonies appear white or blue since there is no other insert DNA here. Iam only trying to do the transformation of E.coli DH5 alpha with pUC 18 using electroporator. so please clear my doubt whether the ransformed colonies will be white or blue at the earliest. |
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By Arun Pandey
on 05-May-2006
Sir i ma doing cloning in Quiagen P Drive Vector After Cloning When We screen the clone our Fragment size decreased Give suggestion I am using DH5 alpha |
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By Diwakar Kumar
on 21-Apr-2006
Dear Sir, |
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By vandana
on 30-Nov-2005
With respect to rajnikant question |
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By Niguse
on 08-Mar-2005
In my blue white screening I found no white colonies but I found the blue colonies has my insert. Is this supposed to happen? |
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By Mary
on 02-Sep-2003
hi, |
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By Rajnikant dixit
on 17-Jul-2002
Dear Sir, |
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