Team:Paris Saclay/Notebook/August/20

From 2014.igem.org

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====CAD cloning in pGEMTeasy====
====CAD cloning in pGEMTeasy====

Revision as of 10:17, 22 August 2014

Contents

Wednesday 20th August

Lab work

Petri dish

by Laetitia

-->10 petri dish using :

- 200mL LB+Agar

-200 µL X-Gal(80mg/mL)

- 200 µL ampi

- 100 µL IPTG


D- Lemon scent

Bacterial culture

by Laetitia

Here are the results of the bacterial culture of yesterday

20 08 bacterial culture.jpg

Bacteria with the pGEMTeasy without the DNA insert are blue and the bacteria with the pGEMTeasy and the DNA insert (PS, GS or LS gene) are white

Then, we have chosen 3 white colonies for each DNA insert and we have launched 3 corresponding liquid cultures :

- 5mL LB + ampiciline (1/1000e)

- 1 white colony

The 9 cultures are incubated at 37°C


by Melanie

Preculture 20ml of DH5a pPSI (apra 1/2000)


CAD PCR

Today we received our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)

so we do a PCR (with GoTaq):

PCR of BBa_K762100
components volumes
DNA of p cola "very small amount"
GoTaq buffer 5X 10μl
DMSO 1.5µl
H2O 23.25µl
MgCl2 4µl
dNTP 1µl
iPS79 5µl
iPS80 5µl
GoTaq polymerase .25µl
PCR cycle
step temperature (°C) time (min)
1 95 2
2 95 .5
3 58 .5
4 72 1.75
5 72 5

CAD cloning in pGEMTeasy

by Sean

protocol

Electrophoresis of pPSI digested by PacI

by Eugene

We made an electrophoresis of pPSI to verify if the digestion made 19/08 has worked. It is usefull before starting the purification of the plasmid.

Migration of pPS1 digested by PacI for the purification

by Huang vu and Laetitia

pPS1 previously digested by PacI is filled inside an agarose gel :

-1g agarose

-1mL TAE

-2 µL BET

The totality of the sample is filled (around 38µL) and we filled 20µl of ladder. Migration 100V

After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification

Digestion of CAD and PMCS5 at 37°C

by Eugene

components volumes
CAD 5 μl
10x buffer fastDigest 1 μl
PacI 0.5 µl
H2O 3.5 µl


components volumes
PMCS5 15 μl
10x buffer fastDigest 2 μl
PacI 0.5 µl
H2O 2.5 µl


B - Construction of the fusion protein (color)=

PCR with Gotaq

22.75 µl --> H2O

10 µl --> 5xbuffer

1 µl --> dNTP

4 µl --> MgCl2

5 µl iPS 83 (Primer)

5 µl iPS 84

0.5 µl --> DNA

1.5 µl --> DMSO

0.25 µl --> Gotaq


PCR cycle

95° 2 min

95° 30 sec

58° 30 sec

72° 1 min 45

72° 5 min

Cloning in pGEMTesay

by Sean

protocol

Back to the calendar

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