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Basic Process for Designing Primers for Cloning

  1. Locate your template
    1. For example, suppose we want to clone 
  2. Design a stretch of DNA that will bind to your template.

Check these sites to see how many nucleotides to add to the 5’ end of restriction sites

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp

USE NEAREST NEIGHBOR MELTING TEMPERATURES (TmNN) FROM

http://www.basic.northwestern.edu/biotools/oligocalc.html

USUALLY AIM FOR TmNN >= 50C

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