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Team:Washington/Magnetosomes/GibsonResults
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==Comparison between pGA and pSB vectors== | ==Comparison between pGA and pSB vectors== | ||
| - | To quantitatively illustrate that better cloning efficiency is achieved by using pGA vectors vs standard pSB vectors, a [https://2011.igem.org/Team:Washington/Protocols/pGA pGA vector assay] was conducted. | + | To quantitatively illustrate that better cloning efficiency is achieved by using pGA vectors vs standard pSB vectors, a [https://2011.igem.org/Team:Washington/Protocols/pGA pGA vector assay] was conducted. For the pGA vector assay, we used a pLacGFP insert from 1C3 and a 1A3 backbone. Both of these had already been previously gel extracted, therefore, we proceed straight to the Gibson Reaction. For the pSB vectors. we started with a pLacGFP insert from 3K3 and 1A3 Backbone. Both the insert and backbone were amplified using PCR (20 ng/uL of DNA), and gel extracted. From this point, we preformed our Gibson Assay. |
| - | Efficiency was calculated by dividing the # of bright colonies by the # of total colonies - # of background colonies. | + | The Gibson products made using our pGA vectors and the standard pSB vectors were plated separately over 6 plates, and the Gibson efficiency was calculated for each plate. Efficiency was calculated by dividing the # of bright colonies by the # of total colonies - # of background colonies. |
Gibson Efficiency = Bright colonies/Total - Background) | Gibson Efficiency = Bright colonies/Total - Background) | ||
Revision as of 00:22, 28 September 2011
Comparison between pGA and pSB vectors
To quantitatively illustrate that better cloning efficiency is achieved by using pGA vectors vs standard pSB vectors, a pGA vector assay was conducted. For the pGA vector assay, we used a pLacGFP insert from 1C3 and a 1A3 backbone. Both of these had already been previously gel extracted, therefore, we proceed straight to the Gibson Reaction. For the pSB vectors. we started with a pLacGFP insert from 3K3 and 1A3 Backbone. Both the insert and backbone were amplified using PCR (20 ng/uL of DNA), and gel extracted. From this point, we preformed our Gibson Assay.
The Gibson products made using our pGA vectors and the standard pSB vectors were plated separately over 6 plates, and the Gibson efficiency was calculated for each plate. Efficiency was calculated by dividing the # of bright colonies by the # of total colonies - # of background colonies. Gibson Efficiency = Bright colonies/Total - Background)
Once the Gibson efficiences were calculated for each set of plates, the average Gibson efficiency was calculated. The average Gibson efficiency of using our pGA vectors was 0.991129032. This value is significantly higher than that of the pSB vectors (efficiency = 0.116395664). Visually, one can see that there are more fluorescent colonies on the pGA plate vs the pSB plate.
Based on the comparison presented, we can be certain that our Gibson Assembly vectors (pGA vectors) are a great choice for gene assemblies!
