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Unformatted text preview: form) - Avoid primer pair complementarities (primer dimers will form and be synthesised preferentially to any other product) - Avoid use of 4 or more consecutive Guanine nucleotides in primer (forms a significantly stable “cruciform” of “guanine tetraplex” 2° structure) Specific considerations for primer design: - Remember to maintain plasmid elements required for protein expression: • In-frame fusions • Stop codons ( reverse primer ) • Shine Dalgarno sequence ( forward primer ) - Always consider restriction sites available in the MCS (multiple cloning site) - When engineering restriction sites at ends of an amplified PCR product, always include a few additional bases after the restriction site ( http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.a sp ) Reference where more details can be found: http://www.mcb.uct.ac.za/pcroptim.htm...
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This note was uploaded on 11/11/2011 for the course BIO 7.012 taught by Professor Lander during the Fall '10 term at MIT.
- Fall '10