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JA- 6/13

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  • after both LR reactions (for JA and AL) from 6/12 incubated at room temp for 24 hours, the protocol was followed exactly
  • the plated transformed e. coli were left in the 30 C incubator at 1:30pm

JA- 6/16

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  • the plated LR transformants from 6/13 yielded no colonies (after incubating constantly since 6/13); the plates were discarded
  • two new transformations were performed following the protocol; the vectors that were transformed were pre-made stock pEXPR Hef1a:BFP and pEXPR TRE-t:eYFP
  • the transformants were plated on LB-Amp plates and incubated at 30C overnight 

 

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JA- 6/17

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  • the transformants from 6/16 yielded ample colonies; two colonies were picked from each plate (one for a MiniPrep and one for a Midiprep), inoculated into separate LB-Amp solutions, and incubated overnight
  • all the primers for the eYFP-BACE2 fusion arrived today
    • these primers unfortunately were designed too long and had undesirably high melting temperatures (approximately Tm = 80C for each primer)
    • nevertheless, PCR was performed using the YFP primers in the hopes that they might still work; either way, the primers were edited in Geneious and re-ordered so that we will have primers with a lower, more ideal melting temperature
    • PCR was not performed on BACE2 because the BACE2 gene first needs to be mini-prepped out of the bacteria it was delivered in; these bacteria have been plated to incubate overnight