Team:WashU/Week4
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The team has finished up the PowerPoint to be presented on the day of the YLC Outreach. In addition, the team made plates as demos for the children using CFP, YFP, GFP and RFP. [Pictures to follow.] | The team has finished up the PowerPoint to be presented on the day of the YLC Outreach. In addition, the team made plates as demos for the children using CFP, YFP, GFP and RFP. [Pictures to follow.] | ||
- | Unfortunately, most of the plates had little to no growth. The vectors used were Kan-resistant while the plates were Amp plates. We discovered that one of the RFP vials was incorrect and not properly bricked. It was only a vector for Kan/Amp resistance and | + | Unfortunately, most of the plates had little to no growth. The vectors used were Kan-resistant while the plates were Amp plates. We discovered that one of the RFP vials was incorrect and not properly bricked. It was only a vector for Kan/Amp resistance and did not contain a functioning RFP. We re-plated that day on Kan plates and obtained successful growth of the GFP and RFP (from a different vial). We concluded from this that the only successful construct was GFP and that RFP was a self-ligated J23100 promoter vector. |
The following day at our meeting with our advisors, we went over the procedures. Andrew discovered that ligase used was 3 years expired so we threw it out and started use of our new ligase from NEB. We were advised to try gel purification after digestion to try to prevent self-ligation. We also feared that the miniprep might have been done poorly for GFP, YFP, and mCherry since they were done by new guys to the task and poorly harvested. Thus, these were grown in liquid culture and re-miniprepped. | The following day at our meeting with our advisors, we went over the procedures. Andrew discovered that ligase used was 3 years expired so we threw it out and started use of our new ligase from NEB. We were advised to try gel purification after digestion to try to prevent self-ligation. We also feared that the miniprep might have been done poorly for GFP, YFP, and mCherry since they were done by new guys to the task and poorly harvested. Thus, these were grown in liquid culture and re-miniprepped. |
Revision as of 21:24, 25 June 2012