Primer Design（Manual and Automated Primer Design for SEQ and PHY Gaps）

General Primer 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 for
Sequencing 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 for
Physical Ends
 Length: 24 to 28 nucleotides
 Tm 62-66oC.
 Pairs with same Tm.
 Unique to template.
 Tm and self-complementarity:
http://www.basic.northwestern.edu/biotools/oligocalc.html

Unique Primer Alignment