"

From 2014.igem.org

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

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 have receive our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)

so we do a PCR (with go taq):

23.25 µl --> H2O

10 µl --> 5xbuffer

1 µl --> dNTP

4 µl --> MgCl2

5 µl iPS 79 (Primer)

5 µl iPS 80

Very small quantity of 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

CAD cloning in pGENTeasy by Sean

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