Part:BBa_K314110:Experience

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Applications of BBa_K314110

The iGEM TEC-CEM collegiate team improved the characterisation of this biobrick. The plasmid pSB1C3 from 2015 kit plate 1 well 1A was transformed into bacterial strain JM109 which is useful for production of single-stranded DNA from M13 or phagemid vectors. [1] After this 50 ml of LB medium without antibiotic were inoculated with a fresh colony and it was grew at 37°C and 250 rpm until de A600=0.05, 50 ul of M13K07 were added and incubated for 60 minutes, kanamycin was added to a final concentration of 70 ul/ug and it was grew ON at 37°C and 250 rpm. The culture was centrifuged at 8000 rpm for 10 minutes and the supernatant was transferred to a new tube and centrifuged again. Supernatant was transferred to a new tube and 0.2 volume of 2.5M NaCl/20%/PEG-8000 was added and incubated for 60 min at 4°C. The phage was recovered by centrifugation at 8000 rpm for 10 min, the supernatant was decanted and the pellet was resuspended with 1.6ml of TE and transferred to micro tubes. 200 of 2.5M NaCl/20%/PEG-8000 was added and it was let sit at room temperature for 5 min, micro tubes were centrifuged at 8000 rpm for 10 minutes, all aqueous phase was removed. Pellet was resuspended with 300 of TE, FCA (phenol/chloroform/isoamylic alcohol) was added and it was let sit for 5 minutes, then centrifuged at 10000 rpm for 5 minutes and the aqueous phase was recovered, 30 of 3M NaOAc was added and 2 Volumes of ethanol at 70%, it was let sit for 15 min, microtubes were centrifuged for 10 min at 12000 rpm and the supernatant was discarded. All the last traces of supernatant were removed and pellet was resuspended with 100 ul of H2O °MB. A kinetic curve was constructed using this protocol for hours 1, 2, and 3.

User Reviews

UNIQad1bbf5c03e90e64-partinfo-00000000-QINU UNIQad1bbf5c03e90e64-partinfo-00000001-QINU

The iGEM TEC-CEM collegiate team worked with this part during summer and it was cocnluded that it is fully functional and it can be applied to the team´s theory of DNAbot production.