I have been doing TA cloning for quite sometime. My sample is giving me only few white colonies. I took two control, the first control is plasmid with insert, which resulted in full plate white colonies. The second control is supercoiled plasmid and I did not find any blue colonies. Apart from X-gal is there any other reason. I want full blue-white colonies, please give your advice.
u will not find any blue colonies if u transform intact plasmids into cells. blue colonies arise when there is no insert into ur pGEMT of T vector that u are using. if the gene is inserted into the vector u shud get white clonies. so it is always a case of probability. not all plasmids would contain insert. i dont understnd the issue..are the white colonies not positive in colony pcr??
Hi Qurshid,
There are two possibilities. First, the ratio of your insert to TA vector may not be optimal. Second, your TA vector made have lost the T over-hang due to repeated freezing and thawing. When you get a new tube of TA vector you should aliquot in into 1-2ul aliquots so you aren't freezing and thawing the entire lot of vector each time.
Good Luck!
Thank You Ms. Sinha and Tom Masi. Ms. Sinha I have transferred supercoiled PTZ vector (control) to check that the blue colonies are forming or not, to my astonishment I did not see formation of any blue colonies. I am just wondering that ligation mix has vector plus insert and self ligated vector, so why the blue colonies are not coming?
I had same problem, than I increased concentration of X-GAL and got few blue colonies and my control (plasmid without insert) plate was ful of blue colonies. Try with different concentration of X-GAL, It may help.
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