| Date | Product | Starting Plasmid | Thermocycler Variables: #cycles / annealing temp / extension time | 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 | |||
| 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 | pEXPR TRE:eYFP | |||
| 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 BACE2 | |||
Q1-BACE2-Q2 (plus betaine) | pEXPR BACE2 | |||
Q1-BACE2-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 | 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) | ||||
| 7/10 | (miRNA generator stuff) | |||
| (see lab notebook 7/11) | ||||
| 7/11 | (miRNA generator stuff) | 30 / 61C / 30s | Gel Picture 7/11 | |
| (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.
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 |