Primers
Given to us as 100 uM, then diluted to 10 uM with TE buffer
PCR
Product | Gene | Starting Plasmid | Forward Primer | Reverse Primer | Tm hi/lo (anneal) |
|---|---|---|---|---|---|
| LacZ (mir-181c) | LacZ | pDONR | iGEM GB 001 F | iGEM GB 002 R | 61/61 (61) |
| LacZ (mir-144) | LacZ | pDONR | iGEM GB 003 F | iGEM GB 004 R | 61/61 (61) |
(CHECK ANNEAL TEMPERATURE ON NEB)
Ran PCR according to protocol on NEB Website
| 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 |
| 35 Cycles | 98°C 60°C 72°C | 10 seconds 30 seconds 60 seconds |
| Final Extension | 72°C | 5 minutes |
| Hold | 4°C |
Electrophoresis Gel
Prepared electrophoresis gel according to protocol: Gel Extraction (includes making gels)
Made 50mL of agarose/TAE/SYBR-Safe solution (enough for one gel).
Loaded lanes as follows:
Lane 1: Hyperladder 1kb (5uL)
Lane 2: CD79A + stop codon (724bp)
Lane 3: CD79A + TCS (718bp)
Lane 4: linker + mKate (768bp)
When loading wells with PCR products, used Parafilm to mix 1uL of 6X Orange dye with 5uL of sample and loaded 5uL of this mixture in a lane. Ran gel at 120V for 30 minutes.
