For best results, back-to-back primers should be designed using our online primer design software, which can be found at: NEBaseChanger.neb.com. For general primer design guidelines, follow the instructions below. Note that both primers do not have to be mutagenic and do not have to be phosphorylated or purified. Substitutions Substitutions are created by designing a mismatch in the center of the mutagenic primer. Include at least 10 nts that are complementary to your plasmid at the 3' end of the primer. To accommodate large mutations (from 7 to 50 per primer), changes should be incorporated at the 5’end of the mutagenic primer. The 5' end of the second primer will begin at the base next to the 5' end of the first primer and proceed in the opposite direction on the complementary strand. This primer can be 100% complementary to the plasmid sequence or can contain mismatches, if desired. The absence of any overlap ensures that exponential (rather than linear) amplification will take place. Deletions Deletions are created by designing primers that flank both sides of the area to be deleted. The two primers should be designed in opposite directions with their 5’ ends adjacent to the area to be deleted. The primers can be 100% complementary to the plasmid sequence or can contain mismatches and/or insertions if desired. Insertions The sequence to be inserted should be added to the 5' end of the mutagenic primer. For insertions >6 nts, the insertion sequence can be split between the two primers. Half of the insert should be added to the 5' end of the forward primer and the other half should be added to the 5' end of the reverse primer. As described for substitutions, there should be at least 10 nts that are complementary to your plasmid on the 3' end of each primer. The maximum size of the insertion is largely dictated by oligo synthesis limitations. Insertions of up to 100 nts (50 nts at the 5’ end of each primer) can routinely be accommodated using this kit. Note: For primers greater than 60 nts long, it is recommended to have them purified by either HPLC or PAGE.