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Team:Paris Saclay/Notebook/August/18
From 2014.igem.org
Contents |
Monday 18th August
Lab work
B - Construction of the fusion protein (color)
PCR of the chromoprotein
by Mélanie
| components | volumes |
|---|---|
| H2O | 27μl |
| 5X Phusion buffer | 10µl |
| dNTP 10mM | 1µl |
| iPS83 | 1µl |
| iPS84 | 1µl |
| DNA | .5µl |
| DMSO | 1.5µl |
| Phusion | .5µl |
| step | temperature (°C) | time (s) |
|---|---|---|
| 1 | 98 | 30 |
| 2 | 98 | 10 |
| 3 | 58 | 30 |
| 4 | 72 | 45 |
| 5 | 72 | 600 |
D - Lemon Scent
PCR of BBa_K517003
By Hoang Vu and Eugène
We realized a PCR to amplify the β-pinene synthase gene.
| components | volumes |
|---|---|
| DNA of BBa_K517003 | 1 μl |
| green goTaq buffer 5X | 10 μl |
| GoTaq enzyme | 0.5 µl |
| H2O | 29.5 µl |
| MgCl2 | 4 µl |
| dNTP | 1µl |
| IPS68bis | 2 µl |
| IPS69bis | 2 µl |
PCR of BBa_K762100
by Sean
Five tubes were prepared. Protocol was modified to match the one used for BBa_K517003 and p cola (PCR conducted on 8th August).
| components | volumes |
|---|---|
| DNA of BBa_K517003 | 1μl |
| green GoTaq buffer 5X | 10μl |
| GoTaq enzyme | 0.5µl |
| H2O | 31.25µl |
| MgCl2 | 4µl |
| dNTP | 1µl |
| IPS66 | 1µl |
| IPS67 | 1µl |
| GoTaq polymerase | .25µl |
PCR of pCola
by Laetitia and Melanie
We realized a PCR to amplify the geraniol synthase gene.
Five tubes were prepared. Protocol was modified to match the one used for pCola (PCR conducted on 8th August).
| components | volumes |
|---|---|
| DNA of p cola | 1μl |
| green GoTaq buffer 5X | 10μl |
| GoTaq enzyme | 0.5µl |
| H2O | 31.25µl |
| MgCl2 | 4µl |
| dNTP | 1µl |
| iPS82 | 1µl |
| iPS91bis | 1µl |
| GoTaq polymerase | .25µl |
Electrophoresis of the 3 PCR
PS : pinene synthase
LS : limonen synthase
GS : geraniol synthase
PS1 - PS2 - PS3 - PS4 - PS5 - PS6 - LS1 - LS2 - LS3 - LS4 - LS5 - GS1 - GS2 - GS3 - GS4 - GS5 - L
We have PCR product !
Ligation of PCR products inside pGEMTeasy
by Laetitia
We used the 3 previously PCR products to perform 3 reaction of ligation. The DNA insert used for each was : PS-6, LS-1 and GS-5
| components | volumes |
|---|---|
| (2X)T4 ligase buffer | 5μL |
| pGEMT easy(50ng) | 1 μl |
| DNA insert : BBa_K517003 digestion product | 3μL |
| Ligase | 1 µl |
| components | volumes |
|---|---|
| (2X)T4 ligase buffer | 5μL |
| pGEMT easy(50ng) | 1 μl |
| DNA insert : BBa_K762100 digestion product | 3μL |
| Ligase | 1 µl |
| components | volumes |
|---|---|
| (2X)T4 ligase buffer | 5μL |
| pGEMT easy(50ng) | 1 μl |
| DNA insert : pCola digestion product | 3μL |
| Ligase | 1 µl |
16°C until afternoon and 4°C during the night
Human Practices
Art & Design
by Terry
Incubation of a preculture of E. Coli with plasmid FNR RBS AmylCP (coding for a blue chromoprotein) in our new M63 medium for 24h.
