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AL - 6/12
added:
1uL of TRE-t (1) @ 5fM
1uL of EYFP @ 5fM
1uL of pDEST 12 @ 10fM
0.5uL of clonase
mixed by pipetting, a few air bubbles in aliquot.
left over night at room temperature
Purpose: practice
JA- 6/12
added:
1uL of Hef1a (1) @ 5fM
1uL of EBFP @ 5fM
1uL of pDEST 12 @ 10fM
0.5uL of clonase
mixed by pipetting, one air bubble in aliquot.
left over night at room temperature
Purpose: practice
JA- 6/13
- after both LR reactions (for JA and AL) from 6/12 incubated at room temp for 24 hours, the Transformation protocol was followed exactly
- the plated transformed e. coli were left in the 30 C incubator at 1:30pm
JA- 6/16
- 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 Transformation 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
JA- 6/17
- 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
- the PCR products of YFP were analyzed on the nano-drop and displayed a concentration of 299.3 ng/uL (I'm not sure how useful this metric is, since it could just be representing the template DNA and not any desired PCR products)
- 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