I have been trying to do cloning with a ~3kbps insert to pET32a vector recently. But never worked for about 5 weeks. Later I try self ligation and this didn't work either. Never got colonies. Last week I did an experiment and hoped this help me to find the problem. There are two steps I suspected might cause my trasformation to fail. 1. Ligation mixture inhibit transformation. 2. There's something wrong with my gel extraction. The following are what I transfromed: a. puc19 : positive control b. circular pET32a : positive control c. run a gel with circular pET32a, cut the band(I choose the brightest part), and then do gel purification with a Qiagen Gel Purification Kit. Transfrom the purified product. (I ran another gel after the gel extraction and the vector was there in circular form. Three bands.) d. add ligase to c. (Here I want to see if ligation mixture inhibit tranformation) e. pET32a digested with NcoI (This I want to check incomplete digestion) f. slef-ligation of e. but gel extraction step is skipped g. cicular pET32a with ligation mixture (same reason as in d) Results: a. lots of colonies b. lots of colonies c. - d. - e. ~50 colonies f. lots of colonies g. lots of colonies So I think there's some problem with gel extraction. And although digestion in e is not complete but the ligation in f did work since it had much more colonies than e. I ask people in my lab but they said I do the same thing with them in gel extraction step. Has anyone here have problem with gel extraction before? Could anybody give me some sugesstions or tell me what might be the mistakes I made? Thank you. --

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