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RF-Cloning can be tricky sometimes, especially when your insert is large or you need to delete a big chunk of sequence from the destination plasmid. Instead of fighting with the extension overlap PCR method, you can often re-purpose your primers for the In-Fusion recombinase method.
http://www.clontech.com/US/Products/Clo … -HD-Liquid
At face value it looks to be a bit more expensive per construct, but a little secret I'm more than happy to share, is that you can cut the recommended reaction size down to 2μl and squeeze 5 times the number of constructs out of a kit (this drops the price to less than $5/reaction!). The one caveat, is that you either need a restriction site between your insertion points, or you need to linearize the plasmid using inverse PCR.
I highly recommend keeping this method in mind as you design your constructs, because it can save a huge amount of headache. And again, the primers that the website spits out can be used for either the PCR or In-Fusion method. In fact, if you know a priori that you're going to use the recombinase, then you can cut down on the size of the plasmid binding side of the primer (15 bases is the magic number), and save some more dollars on primer synthesis.
-Steve
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