Technique / Molecular Biology / Cloning library construction and screening

Blue / white colonies selection problem

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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! Rating: n/a Reply

	By Elhaseen Elesaid on 06-Mar-2008 Hi It mostly because of these few days of incubation during which E.coli already degraded the insert. Rating: n/a Reply

	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 Rating: n/a Reply

	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 tanking u ravendra Rating: n/a Reply

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. Rating: Fair Reply

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. Rating: Fair Reply

	By vijay on 24-Oct-2007 how can maintain the plasmid in the alpha DH5 which has the t7 promoter mostly used for expression Rating: Good Reply

By K.harsha Vardhan on 02-Sep-2007 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. Rating: Very Good Reply

	By Kapil on 04-Jul-2007 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 Rating: Good Reply

	By sanjay on 26-Mar-2007 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 Rating: Good Reply

	By Nguyen Hung Anh on 06-Dec-2006 Can I select the X-gal screening for pGEX4T-3 in DH5alpha /BL21 strains. Thanks so much Rating: n/a Reply

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. Rating: Good Reply

	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 Rating: n/a Reply

	By Diwakar Kumar on 21-Apr-2006 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 Rating: Good Reply

	By vandana on 30-Nov-2005 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. Rating: n/a Reply

	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? Rating: Good Reply

	By Mary on 02-Sep-2003 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. Rating: Good Reply

	By Rajnikant dixit on 17-Jul-2002 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) Rating: Good Reply