| Date | Product | Gene | Starting Plasmid | Forward Primer | Reverse Primer | Thermocycler Variables: # cycles / annealing temp / extension time | Gel Picture | Comments / Deviations from Protocol |
|---|---|---|---|---|---|---|---|---|
| 6/23 | Q2-BACE2-QX | BACE2 | pEXPR BACE2 | iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 30 / 60C / 30s | Picture 6/23 | Diluted primers in water, not TE |
| 6/23 | Q1-eYFP-Q2 | eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP N-term primerF | iGEM 019 eYFP N-term primerR | 30 / 60C / 30s | Picture 6/23 | Diluted primers in water, not TE |
| 6/23 | Q1-BACE2-Q2 | BACE2 | pEXPR BACE2 | iGEM 001-2 BACE2 N-term primerF | iGEM 002-2 BACE2 N-term primerR | 30 / 60C / 30s | Picture 6/23 | Diluted primers in water, not TE |
| 6/23 | Q2-eYFP-QX | eYFP | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR | 30 / 60C / 30s | Picture 6/23 | Diluted primers in water, not TE |
Ran PCRs according to protocol on NEB website: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
Used 20 uL reaction. For plasmids, diluted to concentration 1ng/uL in EB buffer and added 1uL of each dilution to a separate PCR mix. Diluted primers from 100uM stocks to 10uM with TE buffer.
PCR recipe:
| Quantity | Chemical |
| 1 uL | forward primer (10uM) |
| 1 uL | reverse primer (10uM) |
| 1 uL | template DNA (1 ng/uL) |
| 10 uL | Phusion High Fidelity PCR master mix |
| 7 uL | dH2O |
| 20 uL | TOTAL |
Settings for thermocycler (program: PHUSION):
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 30 Cycles | 98°C 60°C 72°C | 5 seconds 30 seconds 30 seconds |
| Final Extension | 72°C | 5 minutes |
| Hold | 4°C |