Blue / white colonies selection problem

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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Hi...
I am cloning a 400bp promoter region in a lacZ minus E.coli strain. I am not supposed to get blue color colonies on the self ligation plate but I am getting it. I changed the plasmid DNA but still I see bluish tinge to the colonies. Its not really too blue also. Can anybody suggest me?
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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).
Or was that the other way around?
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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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Hi
It mostly because of these few days of incubation during which E.coli already degraded the insert.
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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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i am doing homology modelling.i did not know how to generate loop building.can any one suggest me
tanking u
ravendra
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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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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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how can maintain the plasmid in the alpha DH5 which has the t7 promoter mostly used for expression
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Dear sir,
I am using quickchange site directed mutagenesis kit from stratagene for screeing of control mutagenesis. Here the control plasmid having the mutated beta gal gene was back mutated to normal beta gal gene using PCR. Now I had transformed this in DH5a and when plated on LB+amp+IPTG+Xgal, I was getting a lawn of blue coloured growth without any white colonies found. By this I am not able to calculate the Mutagenesis efficiency. can anyone suggest where I am going wrong. Can I presume that the mutation has occured and single colonies need not be obtained. please help me if anyone has used this kit.
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Dear Sir,

If you are spreading X-Galactosidase and the insert disrupts the lacZ gene, then how come some colonies turn blue? Is it because not all the colonies have the insert?

Is this right, colonies that have insert are white. Colonies that do not have insert are blue.

Thank You,
- Kapil
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Dear sir/madam..
i have done this Blue- white screening using E.coli as host.. at first i have both white and blue colonies on my agar which contain IPTG, X-gal and ampicilin..however the next few days all the white colonies have become Blue colour. why does this happen? may i know what the cause? thanks
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Can I select the X-gal screening for pGEX4T-3 in DH5alpha /BL21 strains.
Thanks so much
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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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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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Dear Sir,

I am doing cloning in pUC18 vector, but after transformation I am getting only white colonies even for control experiments. The strain is E.coli DH5 alpha

Thaerefore kindly help me for the same at an earliest possible

your's truly
Diwakar kumar

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With respect to rajnikant question
I too found once all white colonies,and found the culprit behind it the X-gal.It has deteriorated.So use fresh Xgal made in DMF.
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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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hi,

it is mostly that your strain has changed some character,

one easy way is to ask others for a new DH5a strain.

because I met such a question before,so that is my idea.
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Dear Sir,



I am doing blunt end cloning in pUC18 vector, but after transformation I am getting only white colonies even for control experiments. The strain is E.coli DH5 alpha



Thaerefore kindly help me for the same at an earliest possible





Thanking You

Yours sincerely



(Rajnikant)


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