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Basic Process for Designing Primers for Cloning

  1. Locate your template
    1. For example, suppose we want to clone the sequence for pLtetO: tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgacc  
  2. Design a forward primer with the basic structure 5' BUFFER NUCLEOTIDES - RESTRICTION SITE - TEMPLATE ANNEALING PORTION 3'
    1. TEMPLATE ANNEALING PORTION
      1. Since this the forward primer, simply select the 5' nucleotides of the template.  For example, tccctatcagtgatagagattgacatc in the pLtetO example. 
      2. Use a melting temperature calculator to determine the Nearest Neighbor melting temperature (TmNN). There are different ways of calculating melting temperatures but this is often the most accurate. I like to use http://www.basic.northwestern.edu/biotools/oligocalc.html.  For example, tccctatcagtgatagagattgacatc in the pLtetO example has a TmNN of 56.23C. 
      3. Aim for TmNN of >= 50C.  The more bases you choose for this portion, the greater the TmNN will be.  I usually go from 50-53C and not higher, since it is often not necessary and just adds more cost and complexity to oligo synthesis.
      4. That being said, in the pLtetO example above, we selected a longer template annealing portion with a higher TmNN because there is an internal repeat in the pLtetO sequence (see the two bolded and underlined regions which are exactly the same in the sequence tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgacc) * *In order to get specific binding of the primer to the template, we want additional length to the primer. Remember that PCR extension occurs at the 3' end of the primer. Thus, we want to ensure that there is no nonspecific binding at the 3' end of your designed primer. Thus, we designed our template annealing portion to include additional base pairs so that the 3' end of the template annealing portion can only bind in one way to the 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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