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From 2014.igem.org

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

Migration of pPS1 digested by PacI

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, after the purification

Electrophoresis of pPSI

by Eugene

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

Digestion of CAD and PMCS5

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