FastCloneAssist primer design scheme.

A and B, schematics for class 1 primer designing. (A) insert and (B) vector region primer design schemes. Black and gray boxes show selected bases for the forward and reverse primers while the vertical arrows indicate insertion points. Note, box f1c and r1c (A) are exact reverse complements of box f1 and r1 (B), respectively. The size of all other boxes, marked #, is adjusted according to Tm values. C and D, Class 2 primer designing scheme. (C) for deletion primer design, sequences from region box fa & fb should be complementary to box rb and ra respectively and total length of each complementary boxes should be ~8 bp. (D), showing deletion & insertion primer design. The sequence from box i-fa and i-ra should have 16 base pair complementary sequence. Gray arrow marking region of primers sequence and “#” symbol indicates where FastCloneAssist tool adjust length to adjust Tm values.