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

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

• 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

• 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 

• 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
  • Gel of YFP PCR results:
    

• note: the BACE1 stock cell supply is in the "iGEM 2014" box in the -80C freezer on the second floor (215) in the tube with the yellow cap with the side sticker that reads "HsCD00043526"
• note: the BACE2 stock cell supply is in the "iGEM 2014" box in the -80C freezer on the second floor (215) in the tube with the green cap with the side sticker that reads "...somethingsomething76140"

Constructing pEXPR TRE:BACE2-eYFP and pEXPR TRE:eYFP-BACE2

• ran PCR on BACE2 and on eYFP to produce Q1-BACE2-Q2 and Q2-eYFP-QX and Q1-eYFP-Q2 and Q2-BACE2-QX
• order of lanes: ladder, Q2-eYFP-QX, Q1-eYFP-Q2, Q1-BACE2-Q2, Q2-BACE2-QX, (team B-cell receptor-->) 00798, FD798TOS, TCSGo14VP16

Constructing pEXPR Hef1a:BACE1-eYFP and pEXPR Hef1a:eYFP-BACE1

• there were no colonies on the BACE1 plate, so I re-plated the BACE1 cells on a Kan plate

- Troublshooted PCR - considered llowering annealing temp, only doing one pcr at a time

Transfered BACE1 to liquid culture

• added 5mL LB to round bottom tube
• added 5uL of Kan
• picked colony
• placed tube in shaking incubator @ 37 C