Template:BOKU-Vienna/GoldenGate iGEM Parts

Golden Gate iGEM Parts

Send iGEM-Parts easyer and faster to the iGEM-Registry.

Because we worked during our work only with Golden Gate Systems, we had the problem how to send these parts to the iGEM - Registry. But we had an idea. What is if we combine Golden Gate Standard with the FC10 iGEM Standard?


The PCRs

First of all, each Part and also the Vector pSB1C3 has to tranform into GoldenGate Standards with BbsI cut sites.


As you can see in the picture 1 we designed the primer, for each parts in this way that these have the cutsites for the igemregistry and also for BbsI.

The Vector pSB1C3 got also the Cutsite for BbsI in this way, that the BbsI recognition site was removed at the assembly. 

       <a href="pSB1C3iGEM1.jpg" data-title="Primer Design for GoldenGate Igem Registry" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_pSB1C3iGEM1.jpg" alt="Primer Design for GoldenGate Igem Registry">
       </a>

       <a href="gToffGfpSequenceiGEM1.jpg" data-title="Primer Design for GoldenGate Igem Registry" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_gToffGfpSequenceiGEM1.jpg" alt="Primer Design for GoldenGate Igem Registry">
       </a>

And also for each part the primer was designed in this way, that they contain the cutsites for BbsI, EcoRI, NotI, XbaI, SpeI and PstI.


PCR Method

The Method for each PCR was a standard method for Q5 (from NEB)

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Name Volume
Q5 High-Fidelity 2X Master Mix 25 µL
10 µM Forward Primer 2.5 µL
10 µM Reverse Primer 2.5 µL
Template DNA 2 µL
Nuclease free water 18 µL