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Team:Cambridge/Protocols/Primer design
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*The melting temperature for the annealing region of the primers should be calculated; for example on the Finnzymes website. Care must be taken to ensure that any homologous regions in primer tails are also taken into account. Our standard reaction conditions require that primers anneal between 60-65 degrees C. | *The melting temperature for the annealing region of the primers should be calculated; for example on the Finnzymes website. Care must be taken to ensure that any homologous regions in primer tails are also taken into account. Our standard reaction conditions require that primers anneal between 60-65 degrees C. | ||
| - | *It is advisable that these primers end on a G or a C ( | + | *It is advisable that these primers end on a G or a C (GC clamping), as the G-C bond is much stronger as the A-T bond. |
*Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing. | *Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing. | ||
*The tails are usually 20-25 bases long. The annealing region should be at least around 20 bases long, but it should be adjusted in order to accommodate for the other considerations. | *The tails are usually 20-25 bases long. The annealing region should be at least around 20 bases long, but it should be adjusted in order to accommodate for the other considerations. | ||
Revision as of 14:40, 4 August 2011
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Primer Design
Designing primers was necessary for Gibson Assembly, as well as the addition of BioBrick ends to gene sequences short enough for PCR.
Theory
In our experience, primer design can be a lot trickier than it initially seems - several different factors can prevent successful annealing of the primer, and the mispriming likelihood must be consciously minimised as much as possible.
Key considerations
- The melting temperature for the annealing region of the primers should be calculated; for example on the Finnzymes website. Care must be taken to ensure that any homologous regions in primer tails are also taken into account. Our standard reaction conditions require that primers anneal between 60-65 degrees C.
- It is advisable that these primers end on a G or a C (GC clamping), as the G-C bond is much stronger as the A-T bond.
- Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing.
- The tails are usually 20-25 bases long. The annealing region should be at least around 20 bases long, but it should be adjusted in order to accommodate for the other considerations.
Safety
The safety implication of the procedure.
