"
Team:The University of Alberta/4 June 2008
From 2008.igem.org
Continued From Yesterday...
Anthony did a miniprep of our putative Blue Ox + suffix/prefix yesterday evening; David then did a digest of this with EcoRI and PstI to determine if our prefix was correct (i.e. if the digest resulted in two bands, then both the prefix and suffix were made correctly since both sites cut properly; if there was only one band, then the prefix was probably incorrect and the EcoRI did not cut). After imaging the gel, it appeared that there were two bands. However, they were not of the correct sizes that we predicted if both enzymes cut and the Blue Ox insert was excised. Rather, it appeared that the bands were simply the digested and undigested plasmid, meaning that there was probably no insert in the vector to begin with. In hindsight, this makes sense, since we had only one colony on the plate to work with. Ligations never work with 100% efficency so it was very possible (and indeed, true) that this colony had no insert. Perhaps we should have first done colony PCR before proceeding; it would have saved us alot of work. This also renders the glycerol stocks we were making pointless since they do not contain the Blue Ox insert.
Today we are redoing the PCR of the Blue Ox + prefix/suffix and repeating the ligation and transformation. Hopefully this time we will get more growth overnight and have more chances of a colony containing the insert. We will have to do colony PCR this time to make sure we have a positive colony!
