Primers
Given to us Usually given as 100 uM, then diluted 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 | 6164/61 (61) |
| mRFP | mRFP | pL1F_1_S6_S7 | iGEM GB 003 F | iGEM GB 004 R | 61/61 (61) |
...
| 64 (64) |
Ran PCR according to protocol on NEB Website
...
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 35 Cycles | 98°C 60°C64°C 72°C | 10 seconds 30 seconds 60 seconds |
| Final Extension | 72°C | 5 minutes |
| Hold | 4°C |
Ask Brian/Jeremy about changing temps/durations
Electrophoresis Gel
Prepared electrophoresis gel according to protocol
Loaded lanes as follows:
Lane 1: Hyperladder Hyperladder 1kb (5uL)
Lane 2: LacZ (mir-181c)Lane 3: LacZ (mir-144): 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.
...