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SPLiCE: Difference between revisions
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We decided to test the efficacy of SLiCE for the assembly of parts only based on a short flanking homology. This homology is roughly equivalent to that of the biobrick prefix and suffix. This means that a two part assembly, of the insert - such as a gblock - into the standard pSB1C3 vector would only require one Seamless Ligation step. This avoids the standard (and costly!) digestion/ligation steps that usually are required for the biobrick assembly. We tested this approach by ligating the standard J04450 RFP generator to pSB1C3 using 22bp and 21bp of homology (biobrick prefix and suffix :) ) and successfully achieved pink, ligated colonies. | |||
<h4>Materials and methods</h4> | <h4>Materials and methods</h4> | ||
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<h4>Results</h4> | <h4>Results</h4> | ||
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For our first attempt we used the Quick 50ul reaction from above and a higher concentration of insert (10:1), electroporated using the second protocol (see electroporation protocol) using electrocompetent cells kindly donated by the Birkbeck iGEM team. The reaction was a success and we had pink colonies on every plate! | For our first attempt we used the Quick 50ul reaction from above and a higher concentration of insert (10:1), electroporated using the second protocol (see electroporation protocol) using electrocompetent cells kindly donated by the Birkbeck iGEM team. The reaction was a success and we had pink colonies on every plate! | ||
[[File:Sheistheone|thumbnail]] | |||
. For a first dry run we call it a success :) <br> | |||
We obtained 1 colony in the plate containig 50ul of recovered cells after electroporation, 3 colonies on the 100ul and 11 colonies in the 350ul. We used this protocol to successfully make a new part BBa_K1845000 which we submitted to the registry. <br> | We obtained 1 colony in the plate containig 50ul of recovered cells after electroporation, 3 colonies on the 100ul and 11 colonies in the 350ul. We used this protocol to successfully make a new part BBa_K1845000 which we submitted to the registry. <br> | ||
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Following this initial protocol we tweaked the reaction protocols to optimise 10ul reactions and test the use of PEG to speed up the reaction. We used the "Quick SPLiCE" reaction protocol, comparing +/- PEG8000. PEG 8000 is a crowding agent that by reducing the amount of free water in the system (used for the solvation shell of PEG) increases the effective concentration of the reaction components. We used 3ul of the SPLiCE/SLiCE reaction for a total of 30ng of vector DNA (100ng in the 10ul reaction) and plated all the cells (500ul from 50ul of competent cells resuspended in 450ul of 2xTY). Control had water in place of PEG. A picture of the colonies and the colony count is displayed below. | Following this initial protocol we tweaked the reaction protocols to optimise 10ul reactions and test the use of PEG to speed up the reaction. We used the "Quick SPLiCE" reaction protocol, comparing +/- PEG8000. PEG 8000 is a crowding agent that by reducing the amount of free water in the system (used for the solvation shell of PEG) increases the effective concentration of the reaction components. We used 3ul of the SPLiCE/SLiCE reaction for a total of 30ng of vector DNA (100ng in the 10ul reaction) and plated all the cells (500ul from 50ul of competent cells resuspended in 450ul of 2xTY). Control had water in place of PEG. A picture of the colonies and the colony count is displayed below. | ||
[[File:Splicevsslice|thumbnail]] | |||
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