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

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

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