Monday 25th August

Lab work

B - Construction of the fusion protein

plasmid extraction : pGEMTeasy + chromoprotein

by Laetitia

We used the bacteria containing pGEMTeasy + chromoprotein launched by Melanie sunday the 24th for the plasmid extraction, 6 independent cultures which come from 6 independent colonies.

We made a stock of bacteria for each sample (x6).

To extract the plasmid, we used the plasmid DNA purification kit (Macherey-Nagel).

Digestion of pGEMTeasy+chromoproteinby XbaI and PstI

by Laetitia

The goal is to check the presence of the insert inside the plasmid. We digest the 6 samples of purified plasmid.

1h at 37°C

Electrophoresis of the digestion product of pGEMTeasy+chromoprotein

by Laetitia

T : control negatif without enzyme

D : sample digested by XbaI and PstI

T1 - D1 - T2 - D2 - T3 - D3 - E - T4 - D4 - T5 - o - D6

Results : lost of the sample D5 in a hole in the agarose gel

The samples 3, 4 and 6 seems to contain the chromoprotein gene (two bands)

--> We will try another plasmid extraction because the electrophoresis profil doesn't show bands for every sample.

Bacterial culture of DH5a containing pGEMTeasy + chromprotein

by Laetitia

We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction

D - Lemon Scent

Gel electrophoresis of CAD

by Sean

Veirfication of the purification from Friday

Legend

1. ladder 10µl
2. PCR result of CAD

CAD ligation in pPS1

by Mélanie pPSI was previously digest by PacI and dephosphorylated We want to insert the CAD PCR fragment in pPSI to make pPS5.

4H at room temperature

Transformation of DH5a with the ligation

protocol

Culture of bacteria with pPS2 - pPS3 - pPS4

I take some colonies made Friday 22th August

Limonene synthase digestion

Because we don't success to have pPS2 - we try to digest again Limonene synthase in pGMET esay

when I done the electrophoresis to check the digestion, I have a surprise : no digestion.

So we check the pGMETesay plasmid with normaly the inserts (for LS GS and PS)and we saw that there is no insert in the plasmid.

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