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Team:Paris Saclay/Notebook/August/19
From 2014.igem.org
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====Purification of PCR products of limonen synthase==== | ====Purification of PCR products of limonen synthase==== | ||
Revision as of 08:56, 22 August 2014
Contents |
Tuesday 19th August
Lab work
B and D
Electrophoresis with the PpsI plasmide (obtain on thursday) + the chromoprotein PCR: we obtain results as expected
D - Lemon Scent
Digestion of PCR results
by Sean, Hoang Vu, Eugene
| components | volumes |
|---|---|
| PCR purified LS | 15 μl |
| FastDigest green buffer 10X | 2 μl |
| PacI | 1 µl |
| H2O | 2 µl |
| components | volumes |
|---|---|
| PCR purified GS | 15 μl |
| FastDigest green buffer 10X | 2 μl |
| PacI | 1 µl |
| H2O | 2 µl |
| components | volumes |
|---|---|
| PCR purified PS | 15 μl |
| FastDigest green buffer 10X | 2 μl |
| PacI | 1 µl |
| H2O | 2 µl |
| components | volumes |
|---|---|
| Purified pPS1 | 30 μl |
| FastDigest green buffer 10X | 4 μl |
| PacI | 2 µl |
| H2O | 4 µl |
Purification of PCR products of limonen synthase
by Laetitia
We used the PCR products of limonen synthase prepared previously : August 18th
The five samples were pooled in one and then purified using this protocol : protocol
Bacterial transformation
by Laetitia
We used DH5alpha competent bacteria (prepared the 14/08) and they were transformed using the ligation product prepared previously August 18th using this protocol : protocol
We transformed 100µL of competent bacteria and we added the totality of the ligation product (pGEMTeasy containing LS or PS or GS)
Preparation of petri dish
by Terry
-->10 petri dish using :
- 250mL LB+Agar
-250 µL X-Gal(80mg/mL)
- 250 µL ampi
- 125 µL IPTG
-->4 petri dish using :
- 100mL LB+Agar
- 100 µL ampi
Bacterial culture
by Laetitia
After the bacterial transformation, we spread for each strain (LS or PS or GS) :
-100µL on LB-Agar+Amp
-100µL on LB-Agar+Amp+IPTG+X-Gal
-200µL on LB-Agar+Amp+IPTG+X-Gal
-100µL of concentrated bacteria on LB-Agar+Amp+IPTG+X-Gal
37°C at night
