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- Have you checked that PCR program that you are using has an annealing temperature suitable for your ovelapping sequences, an not only for external primers?
- In some cases, I have heard that doing some initial PCR cycles without adding primers to the reaction (with the right annealing temperature, so that the two ovelapping sequences can anneal), and then adding the primers and doing the normal PCR cycles can help.
- Another option in order to increase the specificity could be to do a touch-down PCR.
- I normally use DNA templates that overlap in around 20 bases. If our overlapping sequence is shorter, then it may anneal to other non-desired region in your template, and that's why you get unspecific bands.
- Check that you are putting the right amount of each DNA template and that these have been well purified (i.e. free of PCR inhibitors, etc...)
Good luck!! :-)
#143979 Questions / Help with Fusion PCR
Posted
on 25 October 2012 - 01:57 AM
