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.eduhttp://www.bbioo.com/biotools/oligocalc.html

Unique Primer Alignment

 

 

Automated Primer Design using Primer3

 Web-based or downloadable command-line tool.

 Developed to design primers for PCR.

 Designs primer pairs only.

 Must concatenate adjacent contig ends to design gap primers.

 Calculates Tm, self-complementaity, etc.

 Available at:http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi


Scaffolds of the Sample Bac