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Team:Macquarie Australia/Protocols/GibsonTips
From 2012.igem.org
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<h1><center>Top Ten Tips for Gibson Assembly</center></h1> | <h1><center>Top Ten Tips for Gibson Assembly</center></h1> | ||
<blockquote><ol> | <blockquote><ol> | ||
| - | <li>Consider the overlapping sequence | + | <li><b>Consider the overlapping sequence</b> |
| - | <br>The whole technique revolves around the overlapping sequence, <b>everything</b> can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (T<sub>M</sub>), | + | <br>The whole technique revolves around the overlapping sequence, <b>everything</b> can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (T<sub>M</sub>), if this gets to high then significant problems will arise.</li> |
| - | <li> | + | <li><b>Codon Usage</b> |
| + | <br>Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for <i>E. Coli</i> tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.</li> | ||
| + | <li><b>GC Rich Regions</b> | ||
| + | <br>As touched on in the previous point, GC rich regions make synthesis impossible. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence.</li> | ||
| + | <li> | ||
</ol> | </ol> | ||
Revision as of 10:33, 20 September 2012
Top Ten Tips for Gibson Assembly
- Consider the overlapping sequence
The whole technique revolves around the overlapping sequence, everything can be planned but if this is not correct then the blocks won't anneal. Therefore, the overlapping regions need to be identified and conserved between the two adjacent sections G Blocks. Keep an eye on the Melting Temperature (TM), if this gets to high then significant problems will arise.- Codon Usage
Optimising codons is one of the great things about Gibson Assembly, it allows us to increase the yield of our protein. Unfortunately, in our experience, optimising for E. Coli tends to increase the GC content considerably. As such a lot of the optimisation may need to be reversed to allow for the G Blocks to be synthesised.- GC Rich Regions
As touched on in the previous point, GC rich regions make synthesis impossible. Hairpin loops are formed which prevent elongation. As such, when developing the G Blocks be wary of GC rich regions. If possible, using translation software, change bases in the wobble position to an A or T to keep the integrity of the protein sequence.
