From 2012.igem.org
Team:Technion/3_July_2012
Ilya
Today I gel purified the RS and mCherry PCR products. The new kit seems to give higher yields than the previous one we used. The concentrations for the RS and mCherry are 27 ng/ul and 24.5 ng/ul respectively.
Next, I ran the fusion PCR reaction, and it finally worked! There were a few things different from the previous attempts. We started the whole process from the original plasmids. We used new primers and 30s per 1kb for elongation time calculation like Roee told us. Also, we used GC buffer. Roee also insisted that we gel purify the fragments before the fusion reaction, which was very important as we saw on the gel photo. Finally, we used a high Ta of 65 degrees to match the new primers.
I gel purified the fusion product but didn't measure the concentration yet. I forgot to wait a minute before the elution step but I think we will have enough DNA for the next PCR reaction, for the addition of the prefix and suffix.
Roee suggested we clone the product into a plasmid with a promoter to characterize the induction we get from the riboswitch. We chose BBa_J23119, which is a constitutive promoter.
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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