Our goal for this week was clear: we wanted to have green colonies. Since the last weeks cloning process hasn’t worked out, we had to repeat some steps.
At first, we had to isolate the Plasmid psb1C3-RFP from our red E.coli TOP10 cells. This worked out perfectly fine – at this point I would like to thank miniprep-kits!
We amplified our two previously ordered sequences via PCR and then restricted Part 1, Part 2 and our isolated Plasmid with the enzymes EcoRI, BamHI and PstI. All in all, it worked out very well and we could isolate the DNA-Fragments from our Gel.
While doing this step, a lot of the DNA got lost. This was frustrating, however, there was at least enough DNA for the try to ligate those fragments.
Ligation and transformation worked out this time, anyway there were no green colonies on the plate. Since the colonies were red-edged, we assumed that the plasmid-backbone was ligated – but without the inserts. This is the reason why we decided to sequence our inserts to check if the restriction-sites are correct.
We also discussed in some meetings, if we should try to combine the parts by Gibson-Assembly.