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Team:Paris Saclay/Notebook/July/30
From 2014.igem.org
Contents |
Wednesday 30th July
Lab Work
C - Lemon scent
PCR of BBa_K762100
by Sean
This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.
Oligonucleotides used: iPS66, iPS67
Oligonucleotides were diluted twice from 100µM to 50µM.
Protocol
Add into each PCR tube the following:
| Component | For a total volume of 50μl |
|---|---|
| H2O | 35.5μl |
| Phusion buffer 5X | 10μl |
| dNTPs | 1μl |
| iPS66 | 1μl |
| iPS67 | 1μl |
| BBa_K762100 | 1μl |
| Phusion DNA polymerase | 0.25μl |
Tube was placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
98°C |
1 min |
1 |
| Denaturation | 98°C | 15 s | 25 - 30 |
| Annealing | variable | 55°C | 25 - 30 |
| Extension | 72°C | 45 s | 25-30 |
| Final extension | 72°C | 10 min | 1 |
| Final extension | 8°C | hold | 1 |
