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.


Back to the calendar

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