Team:Paris Saclay/Notebook/August/22

From 2014.igem.org

(Difference between revisions)
(Friday 22nd August)
(Ligation of LS/GS/PS in pPSI)
Line 34: Line 34:
'''(photo)'''
'''(photo)'''
-
==== Ligation of LS/GS/PS in pPSI====
+
==== Ligation of BBa_K517003, BBa_K762100, and p cola in pPSI====
-
For each synthase, put:
+
{| class="wikitable centre" width="25%"
{| class="wikitable centre" width="25%"
-
|+
+
|+ BBa_K517003
|-
|-
! scope=col | components
! scope=col | components
! scope=col | volumes
! scope=col | volumes
|-
|-
-
|synthase
+
|BBa_K517003
 +
|10μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|-
 +
|ligase
 +
|1µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ BBa_K5762100
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|BBa_K762100
 +
|10μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|H<sub>2</sub>O
 +
|5µl
 +
|-
 +
|ligase
 +
|1µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ p cola
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|p cola
|10μl
|10μl
|-
|-

Revision as of 09:34, 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 during 3 hours.

Transformation in competent E.coli DH5 Alpha

After 3 hours, take out the ligation tubes and add 100 µL of competent DH5 Alpha in each tube.

Then procede 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

And finally put it in a incubator at 37°C during 30 minutes

Transformation of pPS3 in DH5 alpha

We add 100 µl of DH5 alpha in the pPS3 ligation tube and we follow the classic protocol of transformation.


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