Team:Cambridge/Protocols/Primer design

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==Primer Design==
==Primer Design==
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Designing primers was necessary for Gibson Assembly and the addition of BioBrick ends to genes ready for submission to the registry.
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Designing primers was necessary for Gibson Assembly, as well as the addition of BioBrick ends to gene sequences short enough for PCR.  
===Theory===
===Theory===
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How it works
 
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===Practice===
 
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Gibson primer has annealing region and tail
 
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The annealing region needs to be selected from the front and the reverse complement of the end of the gene of interest
 
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These regions should be tested on melting temperature calculating websites such as Finnzymes (check for homology at the join and include any homologous regions in the tail in any annealing temperature calculation). Our standard reaction conditions require that primers anneal between 60-65 degrees C. It is also advisable that these primers end on a G or a C (G C clamping) as the G-C bond is much stronger as the A-T bond.
 
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Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing.
 
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The tails are usually 20-25 bases long.
 
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{Standard layout for procedures is to use: 
 
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*'''''<Procedure title - aka what you are doing>'''''
 
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:# <step 1>
 
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:# <step 2>
 
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:#* '''<additional notes/important information regarding the previous step>'''
 
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the text within the < > is what should be written, don't include < > in actual writeup :P
 
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if in doubt see the gel electrophoresis protocol
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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 minimised as much as possible.
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}
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===Key considerations===
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===Safety===
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*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 listed below.
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The safety implication of the procedure.
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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.
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*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.
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*Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing.
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{{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}}

Latest revision as of 20:22, 21 September 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 minimised as much as possible.

Key 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 listed below.
  • 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.

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