cloning methods - something other than the usual (Dec/27/2008 )

hi all..
i have a query... if I have a sequence that encodes a small stretch of amino acids...like about 6 aa, and i want to incorporate this sequence in the middle of an intact gene......is there a specific method to do this or do i follow the standard cloning protocol?????
please let me know asap..........


thanks........

Site directed mutagenesis would be the standard cloning protocol to deal with this situation, especially if you have your starting gene in a plasmid. It is on the large side - 18 bp, but it should work. And the method is the cleanest way of doing things.

The other less elegant method is to use two set of PCR to clone two halves the gene. One set of primer set carrying the sequence changes. Both halves of the gene made have a unique sequence overlap (which has a tm 60 C). Put both halves together and fuse them by PCR. YOu should also add the most terminal primers to further amplify the fused PCR product. The sequence overlaps between each half of the gene, allows them to anneal and extend to produce the complete gene.

Each half of the gene is also engineered to carry a unique restriction site. So once both halves fuse, you cut the final product with both restriction enzymes and ligate that to your vector. Only the final fused product will be able to ligate to the cut vector.

Guard sequence----BamHI------Gene half 1------correction ---overlap


overlap-----gene half 2-----EcoRI -----guard sequence


Fuse by PCR


Guard sequence----BamHI------Gene half 1------overlap-----gene half 2-----EcoRI -----guard sequence

thanks.......will discuss with my supervisor.......

if you do decide to use recombineering, check your sequence first for patches of homology. If it can go wrong it will go wrong. Be on guard of such mistakes

You will need to be able to screen for your the recombined plasmid. PCR is possible (since the insertion is large), and if that 18bp has a restriction site you can use, all the better. You also need a negative test to make sure native plasmid is absent. (again via PCR and restriction sites)

Also, recombinnering isn't clean. Your cell will often have both recombined and native plasmid. Pick the colony that you had identified to contain your recombined plasmid plasmid and grow it up in culture. Then streak to singles colonies. Check several colony to find one which contains only the recombined plasmid. Plasmids which has a high copy number are a pain.