General Primer Design Guidelines
-Note the 5’-3’ direction of the contig.
-Locate primers 100 to 200 bp from the feature.
-Pick primer from ≥ 2x high quality sequence coverage.
-GC Clamp
-Avoid runs of identical nucleotides (e.g.
ATCGACCCCCTAGAC).
-Similar Tm (+/-2℃) for primers in same reaction set.
-Unique to the template.
Designing Primers forSequencing Gaps
-Length: 18 to 21 nucleotides
-Tm 56-60℃
-4+2 Rule:
Tm = (#G + #C) x 4 + (#A + #T) x 2
-Inside the clone
Designing PCR Primers forPhysical Ends
-Length: 24 to 28 nucleotides
-Tm 62-66℃.
-Pairs with same Tm.
-Unique to template.
-Tm and self-complementarity:
http://www.basic.northwestern.edu/biotools/oligocalc.html
Unique Primer Alignment
