Team:Cambridge/Protocols/Primer design

Primer Design

Designing primers was necessary for Gibson Assembly and the addition of BioBrick ends to genes ready for submission to the registry.

Theory

How it works

Practice

Gibson primer has annealing region and tail The annealing region needs to be selected from the front and the reverse complement of the end of the gene of interest 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. Primers should be checked for secondary structure, particularly at the 3' end, since this inhibits annealing. The tails are usually 20-25 bases long.

{Standard layout for procedures is to use:

• <Procedure title - aka what you are doing>

1. <step 1>
2. <step 2>
   • <additional notes/important information regarding the previous step>

the text within the < > is what should be written, don't include < > in actual writeup :P

if in doubt see the gel electrophoresis protocol

}

Safety

The safety implication of the procedure.