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Ligating two inserts? - (Mar/14/2006 )

Hi there. I received a clone that has several problems and my boss wants me to fix the clone before we try to express it. First of all, there is a 3bp insertion right after the start codon and about 100bp downstream of that insertion there is another insertion of 1bp causing a frameshift. After much consideration, I decided to cut out the region where the mutations are and swap in the corrected piece generated by using PCR primers carrying mutations (well in this case I guess we are mutating back to the wildtype). The problem is the gene is very big (~7000bp) and it is very hard to find RE that only cuts once nearing the region of interest. After much thought, I've decided the only way to make it work is to do two separate PCR reactions each fixing one mistake, ligate the two fragments together then put into the vector. And my fragments will look like this after the PCR reaction: 5'-EcoRI-PCR#1-AflIII-3', 5'-AflIII-PCR#2-BspDI-3'.
My question is:
Should I ligate the two PCR fragments first in one ligation reaction then put the ligated segment with vector in a separate ligation reaction or can I throw the two framents and vector all together and just do one ligation? I've never heard of ligations with two inserts so I don't know if it is possible. Although I can't think of any reason it shouldn't work theoretically.

Thanks for all your time.


Yes -- three-way ligations are possible. We do them all the time to bring together flanking pieces of DNA in a suicide vector for use in making deletant strains by allelic exchange.


Even 4 fragments and if you are really careful 5 fragment ligations are possible.


a 3fragment ligation is possible. I prefer to do that on 2steps but one is ok.

Even 4 fragments and if you are really careful 5 fragment ligations are possible.
really?i should came in your lab to do my current %%$&## ligation that doesn't want to occur laugh.gif