Team:Paris Saclay/Notebook/August/22

From 2014.igem.org

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(Transformation in competent E.coli DH5α)
(Transformation of pPS3 in DH5α)
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==== Transformation of pPS3 in DH5α ====
==== Transformation of pPS3 in DH5α ====
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We add 100 µl of DH5α in the pPS3 ligation tube and we follow the classic protocol of transformation.
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We add 100 µl of DH5α in the pPS3 ligation tube and we follow the classic [https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_competent_e.coli_by_CaCl2 protocol] of transformation.
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Revision as of 09:56, 25 August 2014

Contents

Friday 22nd August

Lab work

D - Lemon Scent

pPSI digestion

components volumes
pPSI 40μl
FastDigest green buffer 10X 5μl
PacI 2µl
H2O 2µl

pPSI dephosphorylation

After 2 hours, add 1.5µL of Alkaline Phosphatase (AP) and let it 1 hour.

Then, inactivation of AP at 65°C during 15 minutes.

We've made a electrophoresis to check the digestion.

(photo)

Ligation of BBa_K517003, BBa_K762100, and p cola in pPSI

BBa_K517003
components volumes
BBa_K517003 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl
BBa_K5762100
components volumes
BBa_K762100 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl
p cola
components volumes
p cola 10μl
pPSI 2μl
buffer 2µl
H2O 5µl
ligase 1µl

And let it in the freezer for 3 hours.

Transformation in competent E.coli DH5α

After 3 hours, take out the ligation tubes and add 100 µl of competent DH5α in each tube.

Then proceed to the transformation protocol.

- 20 minutes at 4°C

- 2 minutes 30 seconds at 42°C

- 2 minutes at 4 °C

- Add 0.9 ml of LB into the tubes

Finally put it in a incubator at 37°C for 30 minutes

Transformation of pPS3 in DH5α

We add 100 µl of DH5α in the pPS3 ligation tube and we follow the classic protocol of transformation.


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