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Primers & Sequencing

ELIM Biopharm: Primers & Sequencing

Designing Primers
Choose a forward and reverse primer from a location in the gene or plasmid that is sure to include the portion desired for amplification or sequencing For sequencing, it is desirable if possible to have primers that fall 50-150bp outside your desired region, to ensure that accurate reading occurs for the whole gene (often the first and last ~100bp in the read are very inaccurate). For PCR remember that the sequence portion corresponding to the primers themselves will be amplified also Primers should normally be between 15-30bp in length (around 20bp is ideal)

Desired melting temperatures are generally between 55-65°C As you will see, melting temperature is a function of length and GC content, so it is often difficult to design primers in regions much greater than 50% AT Forward and reverse primers should have the same melting temperature, or with a difference of no more than 3 degrees The annealing temperature used for a pair of primers should be set at 5 degrees below the lower melting point of the primer pair Using a tool like ApE or Geneious makes it easy to select certain sections of a sequence to check for primer features like melting point and GC content IDT's 'Oligo Analyzer' is a great tool to check for primer dimerization, hairpin structures, etc. Use this tool or something like it as a final check to make sure your primers will not be likely to react with themselves or each other around the temperatures they will be active for gene interaction. NCBI Primer Blast is another great tool. It can be used both to help design the primers and to ensure that the primers you choose will not amplify any genomic DNA in a colony based amplification. Primer Blast isn’t perfect. It will often miss off-target products, or predict ones that don’t happen. Special BioBrick considerations

Ordering Primers
We order our primers from ELIM Biopharm. The rest is fairly self-explanatory, but we'll do a walk through when you get here Primers <36bp ordered before 5pm will arrive the next day. Delivery is around 2:15pm.

Primer Dilution (stock preparation)
Once you receive your primers, you need to dilute them; Kosuke does 1/10 dilutions, but iGEM typically uses 1/20 (10μM) dilutions. Typically we create 100-200μl working stocks; it will take a long time to use up that much primer.

Example: 10μL primer stock
190μL qH2O or TE buffer

Sequencing
Two main reasons: (1) after a difficult pcr/gel extraction to ensure the product is correct or (2) after cloning/biobricking to ensure no errors were introduced during PCR.

      

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