Double primer method:
Design mutagenesis primers. The targeted mutation should be included into both primers. The mutation can be as close as 4 bases from the 5-terminus. The mutation should be at least 8 bases from the 3-terminus. At least eight non-overlapping bases should be introduced at the 3-end of each primer. At least one G or C should be at the end of each primer. Design your primers (including the mutations) to have a Tm >=78°C.
Single primer method: Design mutagenesis primer(s). The targeted mutation should be in the middle of the primer. Design your primers (including the mutations) to have a Tm >=78°C.
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