Team:Paris Saclay/Notebook/August/19

From 2014.igem.org

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(→Purification of PCR products of limonen synthase)
(→Lab work)
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====Purification of PCR products of limonen synthase====
====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]
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]
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 dish====
 +
''by Terry''
 +
 +
'''10 petri dish using''' :
 +
 +
- LB+Agar
 +
 +
-IPTG(1M) at 0,5mM
 +
 +
-80µg of X-Gal(80mg/mL)
 +
 +
- Amp (1/1000)
 +
 +
'''4 petri dish using''' :
 +
 +
- LB+Agar
 +
 +
- Amp (1/1000)
 +
 +
====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
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 15:27, 19 August 2014

Contents

Tuesday 19th August

Lab work

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
PCR 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 :

- LB+Agar

-IPTG(1M) at 0,5mM

-80µg of X-Gal(80mg/mL)

- Amp (1/1000)

4 petri dish using :

- LB+Agar

- Amp (1/1000)

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

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/19"