Use: to piece together two regions of a protein/gene, make a small 100 bp region, theoretically mutagenesis although its efficiency is low for this- ask me about it)
For a reference use:
Horton et al., Biotechniques 1990 Gene Splicing by Overlap Extension: Tailor-made genes using the polymerase chain reaction. This will tell you how to design the primers. For troubleshooting I have found stuff on the web under Google Searches for Splicing by Overlap Extension.
Basic idea:
Using 4 primers and two sequential PCR amplifications you can make a mutant anywhere
1) Amplify up the two fragments using one external and one internal primer from a plasmid containing non-mutated DNA region.
The internal primers are complementary to one another and go in opposite directions
1)à ß----(3) (4) à ß----(2)
(Dashes indicate antisense primers ----à)
MY HINTS
2) Set up PCR to amplify piece using primer 1 and 3 and primer 2 and 4. Use as little amount of template that you can get away with 0.1- 0.3ng final DNA in reaction. If you use more there is a decrease in the efficiency of mutation since there are more WT pieces in the mix.
3) Always gel purify the individual pieces 1,3 and 2,4 before setting up the reaction to SOE them together.
4) When adding the pieces add in equimolar amounts to each other, when setting up the PCR do 3 cycles without the external primers and then add the external primers (just pause the program at 94°C). This allows the two pieces to anneal to one another and the polymerase to generate the longer product (the overlapping sequence of each piece acts as a template for the polymerase during these three cycles.