Sewing PCR - (May/13/2009 )
I am trying to put together two PCR molecules using a sewing (fusion) PCR. The lengths of the two PCR molecules are 300 bp and 12 Kb. I would be happy to receive any suggestions if this is a good strategy to go with or not, considering the huge difference in the size of the two molecules (and hence the initial amount of the two templates I need to add). Also since the sewing PCR product is 12.3kb I am not sure if a normal agarose gel is going to separate it from the 12 kb template strand.
You might be better off cloning one fragment into a vector, and then cutting and ligating the second fragment in. Stitching together fragments in the manner you described will give very low yield as it is very inefficient.
I agree with bob1, just because one of your fragment is 12kb and you WILL have hard time amplifying 12kb from PCRed template.
Just in case if you run into the same issue with small fragments, here is my strategy. Wherever there is joint, I attach forward primer of next fragment on reverse primer of the previous fragment and reverse primer of the previous fragment on the forward primer of the next fragment. Amplify both the fragments using these primers and put them in same molar proportion in the PCR tube with forward primer of the previous fragment and reverse primer of the next fragment. You should get choice of your fusion product. Don't forget to check the frame.
Did I make sense? Post if not.
that's exactly what i have planned and it has worked for me previously for other constructs
The problem with this one is that the difference between the final PCR and template isn't enough to separate them on a simple agarose gel (12 and 12.3kb). Also to get an equal molar ratio I have to add 40 times more of the 12kb template than the 300 bp...I am just skeptical but am still going to give it a shot, then moving to bob1's strategy.