| Date | Lane Order | ProductGene | Starting Plasmid | Forward Primer | Reverse Primer | Thermocycler Variables: #cycles / annealing temp / extension time | Gel Picture | ||
|---|---|---|---|---|---|---|---|---|---|
| 6/23ladder (1kb) | | Q2 | -eYFP- | QX | Q1-eYFP-Q2 | Q1-BACE2-Q2 | Q2-BACE2-QXQ2-BACE2-QX | BACE2 | pEXPR BACE2 | iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 30 / 60C / 30s | Gel Picture 6/23 |
| Q1-eYFP-Q2 | eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP N-term primerF | iGEM 019 eYFP N-term primerR | |||||
| Q1-BACE2-Q2BACE2 | pEXPR BACE2iGEM 001-2 BACE2 N-term primerF | iGEM 002-2 BACE2 N-term primerR | |||||||
| Q2-eYFP-QX | eYFP | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR | |||||
| 6/24 | ladder (1kb) | C-term eYFP| C-term BACE2 | N-term eYFP | N-term BACE2 | Q2-BACE2-QXBACE2 | pEXPR BACE2iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 35 / 56.2C / 30s | Gel Picture 6/24 | |||
| Q1-eYFP-Q2eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP N-term primerF | iGEM 019 eYFP N-term primerR|||||||
| Q1-BACE2-Q2 | pEXPR BACE2 | ||||||||
| Q2-eYFP-QX | pEXPR TRE:eYFP | ||||||||
| 6/25 | Q1-BACE2-Q2 | pEXPR BACE2 | 35 / 50.2C / 30s
| Gel Picture 6/25 | |||||
| Q2-BACE2-QX | pEXPR BACE2 | ||||||||
| Q2-BACE2-QX | pEXPR BACE2 | ||||||||
| Q1-eYFP-Q2 | BACE2 | pEXPR BACE2 | iGEM 001-2 BACE2 N-term primerF | iGEM 002-2 BACE2 N-term primerR | Q2-eYFP-QX | eYFP | pEXPR TRE:eYFP | ||
| Q2-eYFP-QX | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR | ||||||
| 6/2526 | Q2-BACE2-QX (plus betaine) | pEXPR BACE2 | 30 / 55C / 1m
ladder (1kb) | N-term BACE2 | C-term BACE2 | N-term eYFP | C-term eYFP
| Gel Picture 6/26 | |||||
Q2-BACE2-QX (plus DMSO) | pEXPR BACE2 | ||||||||
Q1-BACE2-Q2 (plus betaine) | pEXPR BACE2iGEM 016 | ||||||||
Q1-BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | -Q2 (plus DMSO) | pEXPR BACE2 | ||||||
| 6/27 | Q2-BACE1-QX | pEXPR BACE1 | 35 / 50.2C / 30s | Gel Picture 6/ | 2527 | ||||
| Q1Q2-eYFP-Q2QX | pEXPR eYFP | ||||||||
| 6/30 | Q1-eYFP-Q2 | pEXPR eYFP | Gel Picture 6/30 | ||||||
| Q1-BACE1-Q2 | pEXPR BACE1 | ||||||||
| 7/9 | Q2-eYFP-QX (for BACE2) | pEXPR eYFP | 35 / 58C / 1m with both DMSO and Betaine | Gel Picture 7/9 | |||||
| Q1-eYFP-Q2 (for BACE2) | pEXPR eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP N-term primerF | iGEM 019 eYFP N-term primerR | Q1-BACE2-Q2 | BACE2 | pEXPR BACE2 | iGEM 001-2 BACE2 N-term primerF | iGEM 002-2 BACE2 N-term primerR |
| Q2-eYFP-QX (for BACE1) | pEXPR eYFP | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR||||||
| Q2-BACE2-QX | pEXPR BACE2 | ||||||||
| Q1-BACE2-QX | pEXPR BACE2 | ||||||||
| Q1-BACE1-QX | pEXPR BACE1 | ||||||||
| 7/10 | (miRNA generator stuff) | 35 / 58C / 1m with both DMSO and Betaine | Gel Picture 7/10 | ||||||
| (20 PCR products in all) | |||||||||
| 7/11 | (miRNA generator stuff) | 30 / 61C / 30s | Gel Picture 7/11 | ||||||
| (12 PCR products in all) | |||||||||
| 7/16 | (miRNA generator stuff) | 30 / 63C / 30s | |||||||
| (12 PCR products in all) |
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.
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