IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Gold Bioremediation/2010/09/24
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PCR verification of pgolS and golB + Terminator was done using VF2/VR primers in 20 uL reactions. Culture broth was used instead of minipreps for these.
A gel electrophoresis yielded bands for all of the culture PCRs.
Innoculations were made for pgolS + E0420 + pSBIT3, pgolS + E0430 + Psbit3, and golS + Terminator + pSBIT3. All were incubated overnight in a roller at 37 °C.
The PCR purifications of gesA, gesC, golS with BsaI, and golb with BsaI was completed yielding concentrations between 17.1 ng/uL and 86.6 ng/uL.
The golS with BsaI and golb with BsaI PCR purfications were digested as well in 50 uL digestions. Both of the digestions were then ligated to lamb and then transformed using heat shock and were shook for 1 hour and then plated and incubated overnight in the 37 °C room.
GesA, gesB, and gesC were re-PCRed in 20 uL reactions.