Ligation of PCR fragments - (Sep/22/2013 )

i didnt know exactly how much dna i put, but i used 1uL, when i get smearing, i diluted the template 100x,1000x,10kx and 100kx time, but still smearing.

Hi!

I don't know if you have already solved your problem, but for my it also seems that you are making too many purifications, ligations and so on.

When I have had to fuse 3 fragments A, B and C, I usually amplify the three of them separately by PCR, clean the PCR with a column if I get a single band (if not, I do a gel-purification by freeze and squeeze).

Then I fuse A+B in a recombinant PCR and B+C in another recombinant PCR. 

Finally, either I join A+B with B+C using a restriction site if it is available in B, or I make a third recombinant PCR in which the templates would be both A+B and B+C and the final product should be A+B+C. In the first case I would directly clone A+B and B+C into the vector by making a triple ligation. In the second case, I would clean the PCR and I would proceed with the cloning.

For me it's much easier and I have done it many times (with success!). Most probably, you will only need to design long primers containing tails for the overlapping ;)

Hope it helps and hope it works!