Team:The University of Alberta/16 June 2008

DBN meeting today @ 3PM in Comp-Sci 2-49 (?). The NINT team is presenting so go and show your support! There will be free pizza and door prizes supplied by NINT*

Troubleshooting Day
We have been getting alot of bad results (or no results at all) in the lab lately - our gel purifications have had very low yields, the Westerns have no been working, our transformants wont grow, etc. We have decided that the next few days will be dedicated to troubleshooting so we can figure out what's going on, or what isn't going on. Troubleshooting tasks have been divided up thusly: Jason This will let us know two things: if the BioBricks we currently have in J61003 are actually the biobricks we want and if the original genes we were given in pUC57 are actually the reporter genes they are purproted to be. This could help explain why our Purple Russian colonies aren't purple, and why our PCR results are always so sketchy. Our transformants havent been growing for some reason so we are goign to try and transform them yet again. We will also try tansforming them into the commercially prepared cells to compare Tom and Winnie This should help us understand what's going on with our butanol stuff (why they dont seem to be growing in I0500, etc) Chris Our westerns have been totally blank and we dont know why. Redoing them, in conjuction with Tom and Winnie's stuff above, may shed some light into why they have not been working. Saima
 * Digest reporter genes from pUC57
 * gel purify
 * sequence
 * Digest biobricks from J61003
 * gel purify
 * sequence
 * Retransform biobrick ligations into XL-10 Gold competent cells
 * Set up O/Ns of butanol stuff for protein expression
 * Use Ni-NTA columns to purify
 * Run on SDS-PAGE gel to confirm
 * Sequence, PCR, digest, confirm butanol biobricks
 * Redo Westerns
 * Make 3 gels - one from each of the 3 sources of agarose in the lab - then run a standard and gel purify. This will let us know if the agarose we have been using is the cause of the low yields from gel purifications.

Troubleshooting Results
Saima After running the 4 gels, the one made from the '420' agarose appeared to have the sharpest bands. The bands from each were gel extracted and the concentrations were recorded: It would seem that the type of agarose we've been using hasn't affected the recovery at all, so the problem must be at a step further along in the procedure. One possibility was that we were losing the DNA in the waste that we normally discard. To test this, we tried to re-elute any DNA that might be in the waste. The results were as follows: Low concentrations such as this would be expected in the waste since there shouldnt be any DNA in there to begin with; if we were losing the DNA in there, however, we would expect to see higher numbers. Another possibility is that the DNA is still bound to the column and not eluting. To test this we added an additional 50ul of EB, let the columns sit for 5 minutes and then centrifuge. Results were: This time, there was an equal amount of DNA (give or take) eluted from the columns as the first time. This might indicate that the DNA is not eluting off of the column for some reason. Tomorrow, we will try columns from different kits to see if they make a difference.
 * '375' Agarose: 2.7ng/ul  0.8
 * '420' Agarose: 5.8ng/ul  0.9
 * 'iGEM' Agarose: 1.9ng/ul 0.4
 * '375' Agarose: 0.8ng/ul
 * '420' Agarose 0.9ng/ul
 * 'iGEM' Agarose: 0.4ng/ul
 * '375' Agarose: 4.8
 * '420' Agarose: 3.8
 * 'iGEM' Agarose: 6.5