From 2012.igem.org
Team:Technion/20_June_2012
Ilya
Yesterday, Asaf and I ran the gradient PCR reaction to try the fusion again. We also changed the buffer from HF to GC.
Today, I ran the resulting PCR products on a gel. In all the temperatures we got a strong unexpected band at ~600bp and a weak expected band at ~1200bp.
Roee suspects some sort of problem with the primers. However, before we design new primers, I suggested revising the PCR program. For some reason, Sharon told us to have a 2 min step at 37 degrees for the first 10 cycles of the PCR for annealing. We are going to try to do the fusion PCR with the regular PCR program, without the steps at 37 degrees. Another variation of the protocol we are going to try, is starting the reaction without primers, allowing the overlaping areas to fuse and enrich the desired template. Next, we will add the primers with more DNTPs to do the amplification of the desired product. In those two configurations we are going to increase the elongation time like Roee told us to do.
Once we run the results of those two PCR reactions, we will think again what to do.
Maybe we will need to redesign the sense primer for the fusion. Also, we might need a new primer for the reaction which adds the prefix and suffix since we never managed to get a result with that primer.
Another action to consider, is sequencing the pUC19 plasmid with RS to see that we are really working with the DNA we think we are working with.
Finally, we might also gel purify the best bands we have at ~1200bp for further experiments.
On a different note, we met the genomecompiler folks today and gave them more feedback about their software. They also showed us some of the new features of their latest updated release. Soon, they will order our sequence for the RS+T3 RNAP.
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
Today we plated the bacterias (with T7RANP and BBA_0015)on medium antibiotics (LB+amp)in preparation for miniprep.
|
"
