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
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)