Ran PCRs for the following samples:
| Date | ProductGene | Starting Plasmid | Forward Primer | Reverse Primer | Tm hi/lo (anneal) | Thermocycler Variables: #cycles / annealing temp / extension time | Link to Gel Picture | ||
|---|---|---|---|---|---|---|---|---|---|
| 6/23 | Q2-BACE2-QX | pEXPR BACE2 | 30 / 60C / 30s | Gel Picture 6/23 | |||||
| Q1-eYFP-Q2 | pEXPR TRE:eYFP | ||||||||
| Q1-BACE2-Q2 | pEXPR BACE2 | iGEM 016 BACE2 primer forward | iGEM 017 BACE2 primer reverse | (60) | Picture 6/23 | ||||
| Q2-eYFP-QX | pEXPR TRE:eYFP | ||||||||
| 6/24 | Q2-BACE2-QX | pEXPR BACE2 | 35 / 56.2C / 30s | Gel Picture 6/24 | |||||
| Q1-eYFP-Q2 | pEXPR TRE:eYFP | ||||||||
| 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 | 6/23 | Q1-eYFP-QX | eYFP | pEXPR TRE:eYFP | iGEM 018 eYFP primer forward | iGEM 019 eYFP primer reverse | (60) | Picture 6/23 | |
| Q2-eYFP-QX | pEXPR TRE:eYFP | ||||||||
| 6/26 | Q2-BACE2-QX (plus betaine) | pEXPR BACE2 | 30 / 55C / 1m
| Gel Picture 6/26 | |||||
Q2-BACE2-QX (plus DMSO) | pEXPR BACE26/23 | ||||||||
Q1-BACE2-Q2 BACE2(plus betaine) | pEXPR BACE2iGEM 001 | ||||||||
Q1- 2BACE2 _Primer_F | iGEM 002-2 BACE2_Primer_R | (60) | Picture 6/23 | 6/23-Q2 (plus DMSO) | pEXPR BACE2 | ||||
| 6/27 | Q2-BACE1-QX | pEXPR BACE1 | Gel Picture 6/27 | ||||||
| Q2-eYFP-QX | 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 | pEXPR TRE:eYFP | iGEM 003-2 YFP_Primer_F | iGEM 004-2 YFP_Primer_R | (60) | Picture 6/23Gel 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.
...
| 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 |