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Team:Cambridge/Diary/Week 11
From 2012.igem.org
| Week: | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
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Monday (3/9)
The plates from yesterday showed that there is no significant difference between the competency of our cells, or our master mixes. However PJ obtained a >10x greater efficiency.
We decided to rule out a problem with the plates. He'd been pre-warming them, I hadn't. I assembled and transformed again, using 2x our plates and 2x his plates, one warm and one straight from the fridge.
Tuesday (4/9)
Plates from yesterday showed no difference between his plates and ours.
Paul, another member of the Haseloff lab, suggested that he observe me assemble and transform, but did not detect any gross incompetency. We thought that the problem might be the PCR machine - it takes a little while to load ours, whereas theirs is a hotblock that can be preheated, minimizing the time the T5 exo has to digest our fragments.
New control fragments were obtained, some with different "minelute" gel extraction kits. One assembly was set up with mineluted control, the other with normally extracted control. Additionally, a reaction was set up to assemble our 4-part ratiometric construct. We used the Haseloff hotblock PCR machine to assemble. Transformation was done with our competent cells, and they were plated.
Wednesday 5/9
Only one of the control plates from yesterday (the minelute control) has colonies on it, and still a low number. The other has none on it at all. The ratiometric construct plate has a couple of colonies on it, but their lack of fluorescence implies that they are not what we want.
A gel was run to check the control fragments. (5ul of each 10ul elution). No significant differences observed between fragments, all were visible and correctly sized.
