| Date | Lane Order | ProductGene | Starting Plasmid | Forward Primer | Reverse Primer | Thermocycler Variables: #cycles / annealing temp / extension time | Gel Picture | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 6/23ladder (1kb) | | Q2- | eYFPBACE2-QX | pEXPR BACE2 | 30 / 60C / 30s | Gel Picture 6/23 | ||||||
| Q1-eYFP-Q2 | pEXPR TRE:eYFP | ||||||||||
| Q1-BACE2-Q2 | |pEXPR BACE2 | ||||||||||
| Q2- | BACE2eYFP-QX | pEXPR TRE:eYFP | |||||||||
| 6/24 | Q2-BACE2-QX | BACE2 | pEXPR BACE2 | iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 30 / 60C 35 / 56.2C / 30s | Gel Picture 6/2324 | ||||
| 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 | ||||||||
| 6/24 | ladder (1kb) | C-term eYFP| C-term BACE2 | N-term eYFP | N-term BACE2 | 26 | Q2-BACE2-QX BACE2(plus betaine) | pEXPR BACE2 | iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 35 / 56.2C / 30s | Picture 6/24 | 30 / 55C / 1m
| Gel Picture 6/26 | |
Q2-BACE2-QX (plus DMSO) | pEXPR BACE2 | ||||||||||
Q1-BACE2-Q2 (plus betaine) | pEXPR BACE2 | Q1-eYFP-Q2 | eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP N-term primerF | iGEM 019 eYFP N-term primerR | |||||
Q1-BACE2-Q2 BACE2(plus DMSO) | pEXPR BACE2 | iGEM 001-2 BACE2 N-term primerF | iGEM 002-2 BACE2 N-term primerR|||||||||
| 6/27 | Q2-BACE1-QX | pEXPR BACE1 | Gel Picture 6/27 | ||||||||
| Q2-eYFP-QX | pEXPR eYFP | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR | |||||||
| 6/25 | ladder (1kb) | N-term BACE2 | C-term BACE2 | N-term eYFP | C-term eYFP
| Q2-BACE2-QX | BACE2 | pEXPR BACE2 | iGEM 016 BACE2 C-term primerF | iGEM 017 BACE2 C-term primerR | 35 / 50.2C / 30s | ||||
| Q1-eYFP-Q2 | 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 | eYFP | pEXPR TRE:eYFP | iGEM 003-2 YFP C-term primerF | iGEM 004-2 YFP C-term primerR|||
| 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 | ||||||||||
| Q2-eYFP-QX (for BACE1) | pEXPR eYFP | ||||||||||
| 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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