| Primer Guidelines |
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In a cycle sequencing reaction you can only read one strand at a time:
Use only ONE PRIMER per reaction. |
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Always write the sequence of your primer 5’ to 3’:
The polymerase will extend from the 3’ end |
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The Tm for your oligo should be between 55°C – 75°C |
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The length should be between 18-25 nucleotides |
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Avoid primer-dimers that form stable bonds |
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Primers should be at least 50 base pairs from the target sequence but no more than 300 nucleotides away. Sometimes a sequence reaction can start immediately after a primer, but the most accurate trace data usually begins between 50-150 bps into the read |
