Difference between revisions of "Part:BBa K1807000:Experience"
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iGEM York 2015 used this part as an expression vector and assembled multiple IPTG-inducible protein generators. This part collection was assembled with colony PCR products that were gel extracted and with synthesized dsDNA fragments (G Blocks). | iGEM York 2015 used this part as an expression vector and assembled multiple IPTG-inducible protein generators. This part collection was assembled with colony PCR products that were gel extracted and with synthesized dsDNA fragments (G Blocks). | ||
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| + | == User Reviews == | ||
Revision as of 18:14, 18 September 2015
Above is a photograph of the BBa_K1807000 Gibson Assembly Reaction transformation. Escherichia coli NEB 5 alpha cells were spread onto LB Agar Plates containing Chloramphenicol 34ng/mL, IPTG ng/mL, Xgal ng/mL.
For the Gibson Assembly Reaction we mixed 100ng of PCR-linearized (provided with the distribution) and EcoRI-digested pSB1C3 vector backbone together with 3 times the molar ratio of our Adapter BioBrick G Block dsDNA. We incubated the reaction at 50 degrees Celsius for an hour and then used 2uL (out of 20uL total) in a standard heat-shock transformation of chemically competent cells.
Result: We did not observe white colonies on the plate - thus we estimated the Gibson Assembly efficiency for this reaction to be 100%.
We have sequenced single three colonies (clones) from the plate.
Applications of BBa_K1807000
iGEM York 2015 used this part as an expression vector and assembled multiple IPTG-inducible protein generators. This part collection was assembled with colony PCR products that were gel extracted and with synthesized dsDNA fragments (G Blocks).