I am new to cloning and I am trying to clone a 5 Kb promoter sequence into the pGEM-T vector. As the size of the vector is around 3 Kb, is it possible to clone the fragment? Or can anyone suggest any other effective TA cloning vector for this fragment?
HI, yes I have managed to clone up to 7kb into pGEM-T-Easy. Do an overnight ligation at 4C (instead of the quicker room temperature option) and make sure your competent cells have a high competency. You may not get a lot of white colonies, but 5 kb is definitely possible
Thank you Renate! I am going with the JM109 competent cells, which comes along with my pGEM-T kit. I hope it works.
The key point is to accept here is that the "vector" should be the one that should do the work of "carrying" the cloned-to-be material...if your vector is way smaller than your fragment the efficiency drops...however, it is not impossible to get positive clones.
As many others, I have done this in the past by using a "fast" ligation with Ligafast (promega) with 7.5 Kb fragment...but instead using 5' as suggested I extended it up to 30' (no more than 1 or 2 hs this with this buffer otherwise you get lots of self-ligating vector as well as concatenatemers containing clones). This gave me a few colonies plating the standard 100 uL. Then, I span down the rest of cells and plated them on a secondary plate...I got at least ~5x colonies.. and amongst them I always managed to get a positive clones. In fact, I managed to even get right orientation...which, good for you, it's not an issue.
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