Team:Paris Saclay/Notebook/August/19
From 2014.igem.org
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==Lab work== | ==Lab work== | ||
- | === | + | ===Chromoprotein + Lemon scent=== |
+ | Electrophoresis with the PpsI plasmide (obtain on thursday) + the chromoprotein PCR: | ||
+ | we obtain results as expected | ||
+ | |||
+ | #ladder 10µl | ||
+ | #pPSI 1.2µl | ||
+ | #pPSI 1.2µl | ||
+ | #pPSI 1.2µl | ||
+ | #pPSI 1.2µl | ||
+ | #pPSI 1.2µl | ||
+ | #p cola 1.2µl | ||
+ | #PCR chromoprotein 2µl | ||
+ | |||
+ | [[File:Paris_Saclay_140819.jpg]] | ||
+ | |||
+ | ===Lemon Scent=== | ||
==== Digestion of PCR results ==== | ==== Digestion of PCR results ==== | ||
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|2 µl | |2 µl | ||
|} | |} | ||
- | |||
{| class="wikitable centre" width="50%" | {| class="wikitable centre" width="50%" | ||
|+ | |+ | ||
- | |- | + | |-''Italic text'' |
! scope=col | components | ! scope=col | components | ||
! scope=col | volumes | ! scope=col | volumes | ||
|- | |- | ||
- | | | + | |Purified pPS1 |
|30 μl | |30 μl | ||
|- | |- | ||
Line 84: | Line 98: | ||
|4 µl | |4 µl | ||
|} | |} | ||
+ | |||
+ | ====Purification of PCR products of limonen synthase==== | ||
+ | ''by Laetitia'' | ||
+ | |||
+ | We used the PCR products of limonen synthase prepared previously : [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/18 August 18th] | ||
+ | |||
+ | The five samples were pooled in one and then purified using this protocol : [https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up protocol] | ||
+ | |||
+ | ====Bacterial transformation==== | ||
+ | ''by Laetitia'' | ||
+ | |||
+ | We used DH5alpha competent bacteria (prepared the 14/08) and they were transformed using the ligation product prepared previously [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/18 August 18th] using this protocol : [https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_supercompetent_Ecoli_cells_with_CaCl2 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 dishes==== | ||
+ | ''by Terry'' | ||
+ | |||
+ | {| class="wikitable centre" width="25%" | ||
+ | |+ 10 dishes with: | ||
+ | |- | ||
+ | ! scope=col | Antibiotic | ||
+ | ! scope=col | Concentration | ||
+ | |- | ||
+ | |XGal | ||
+ | |80μg/ml | ||
+ | |- | ||
+ | |IPTG | ||
+ | |2*10<sup>-3</sup> | ||
+ | |- | ||
+ | |Ampicillin | ||
+ | |10<sup>-3</sup> | ||
+ | |} | ||
+ | |||
+ | {| class="wikitable centre" width="25%" | ||
+ | |+ 4 dishes with: | ||
+ | |- | ||
+ | ! scope=col | Antibiotic | ||
+ | ! scope=col | Concentration | ||
+ | |- | ||
+ | |Ampicillin | ||
+ | |4*10<sup>-4</sup> | ||
+ | |} | ||
+ | |||
+ | ====Bacterial culture==== | ||
+ | ''by Laetitia'' | ||
+ | |||
+ | After the bacterial transformation, we spread for each strain (LS or PS or GS): | ||
+ | |||
+ | {| class="wikitable centre" width="25%" style="text-align:center" | ||
+ | |+ | ||
+ | |- | ||
+ | ! scope=col | dish type | ||
+ | ! scope=col | 100µl | ||
+ | ! scope=col | 200µl | ||
+ | ! scope=col | concentrate | ||
+ | ! scope=col | control | ||
+ | |- | ||
+ | |Ampicillin | ||
+ | |X | ||
+ | | | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | |Ampicillin+IPTG+XGal | ||
+ | |X | ||
+ | |X | ||
+ | |X | ||
+ | |X | ||
+ | |} | ||
+ | |||
+ | We leave the dishes overnight at 37°C. | ||
- | == | + | ==Photo of the Day== |
- | + | [[File:Paris Saclay 19_august.jpg|600px|center]] | |
- | + | ||
+ | '''Members present:''' | ||
+ | * Instructors and advisors: Alice, Solenne and Sylvie. | ||
+ | * Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} |
Latest revision as of 15:02, 14 October 2014
Contents |
Tuesday 19th August
Lab work
Chromoprotein + Lemon scent
Electrophoresis with the PpsI plasmide (obtain on thursday) + the chromoprotein PCR: we obtain results as expected
- ladder 10µl
- pPSI 1.2µl
- pPSI 1.2µl
- pPSI 1.2µl
- pPSI 1.2µl
- pPSI 1.2µl
- p cola 1.2µl
- PCR chromoprotein 2µl
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 dishes
by Terry
Antibiotic | Concentration |
---|---|
XGal | 80μg/ml |
IPTG | 2*10-3 |
Ampicillin | 10-3 |
Antibiotic | Concentration |
---|---|
Ampicillin | 4*10-4 |
Bacterial culture
by Laetitia
After the bacterial transformation, we spread for each strain (LS or PS or GS):
dish type | 100µl | 200µl | concentrate | control |
---|---|---|---|---|
Ampicillin | X | |||
Ampicillin+IPTG+XGal | X | X | X | X |
We leave the dishes overnight at 37°C.
Photo of the Day
Members present:
- Instructors and advisors: Alice, Solenne and Sylvie.
- Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.