Page History

- Troubleshooted PCR - considered lowering annealing temp, only doing one pcr at a time

Transferred BACE1 to liquid culture

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

PCR on BACE2 and eYFP

performed for both constructs (N and C term) of BACE2 and eYFP

• 6.4 uL H2O
• 1uL forward primer
• 1uL reverse primer
• 1uL template DNA @1ng/uL
• 0.6uL DMSO
• 10uL 2X Phusion master mix

Annealing temperature was determined from the NEB calculator to be 58C and 59C for the two constructs. The lower temperature was chosen to do all at the same time. Since this mix includes 3% DMSO, the annealing temp was lowered by 0.6C per 1% DMSO to 56.2C

Gel

Hyperladder 1kb | C eYFP | C BACE2 | N eYFP | N BACE2 @120V