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From 2011.igem.org

Team:Baltimore/Notebook

Hours

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Travis- 3PM Pat -2pm

On Thursday, we decided that because the plasmid preps of the mutagenized pET-Taq plasmid (to check for the presence or absence of the PstI site) gave no yield of plasmid DNA, we should regrow the cultures until they were visibly cloudy before isolating the plasmid. Lisa grew 18 LB+amp cultures in her lab overnight at 37C (3 from the 3B plate and 15 from the 4A plate). Of these, only six of the 18 cultures had any bacterial growth at all in LB+amp broth. When DNA was purified from the 6 cultures that showed some growth, there was no yield of plasmid DNA and only cellular RNA was visible. Therefore, there appears to be a problem with the bacteria on the plates.

At this point, it probably makes sense to re-transform the original mutagenesis products into competent bacteria. Also, we should continue to troubleshoot the PCR reaction with the biobrick prefix and suffix primers using the pET-Taq plasmid as a positive control so that once we get more colonies on the plate we can repeat the colony PCR with the biobrick prefix and suffix primers.