Primers
Usually given as 100 uM, so dilute to 10 uM with TE buffer
PCR
Product | Gene | Starting Plasmid | Forward Primer | Reverse Primer | Tm hi/lo (anneal) |
|---|---|---|---|---|---|
| mRFP | mRFP | pL1F_1_S6_S7 | iGEM GB 001 F | iGEM GB 002 R | 64/64 (64) |
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 64°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
Loaded lanes as follows:
Lane 1: Hyperladder 1kb (5uL)
Lane 2: mRFP cassette
Lane 3: P5-N R
Lane 4: P5-N C
Include lengths of dna
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.
(insert pictures of gel here)