Team:The University of Alberta/4 June 2008

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 too close together to be the bands 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!

EDIT Turns out that the digest did work afterall. Jason ran the digests out on another gel with a ladder and we got two very distinct bands, each the size of the predicted digestion products. This means our primers were correct! I guess the gel that we ran last night simply wasn't run long enough and the bands just looked too close together. Since we didnt run the gel with a ladder, we had no way of knowing that the gel needed to be run further to get a better resolution. So... Good News : The EcoRI site in the prefix for Blue Ox works and we can just cut and paste it into a BioBrick vector to finalize it. Bad News : We didnt know that everything worked until AFTER we had thrown out the glycerol stocks we were making. This means we had to set up an O/N culture from what remained of the transformation we did on Monday. We're also redoing the PCR/ligation/transformation again just so we can have a plate full of colonies if we ever need to do something with the Blue Ox "pre-brick". -Cwk 22:53, 4 June 2008 (UTC)

Lab Tip Of The Day
Always use a ladder when you are running a gel, even if you dont care what the size of the bands are. Also, don't throw stuff away unless you are absolutely, positively 100% sure that you're not going to need it in the future.