"

From 2013.igem.org

{ "date" : "2013-07-31", "author" : "thomas", "title" : "pSpac + GFP cloning!!!", "content" : "Today I started a new cloning in order to obtain GFP in the vector pSBBs0K-Pspac (BBa_K823026). This construct will be useful to Emil in order to characterize Pspac at difference concentration of inducer (IPTG). I started performing a PCR on the GFP following the Phusion PCR protocol. I used BB_fwd and BB_rev primers to obtain an amplification of the insert. This because the GFP backbone (BBa_E0840) had the same antibiotic resisistance of our destination vector BBa_K823026 (Amp). I obtain a PCR product with a concentration of 146 ng/µl, confirmed by electrophoresis analysis.

I continued then with the restriction digestions following this protocol. I digested BBa_K823026 using SpeI and PstI, and PCR product with XbaI and PstI.
The two digestion products were then purified and quantified: GFP PCR 60 ng/µl, BBa_K823026 19 ng/µl. The last step was then the ligation that was performed following this protocol. The ligation products were then plated on CM plates. ", "tags" : "pSpac-GFP" }