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From 2014.igem.org

Contents 1 LabWork 1.1 B- Construction of the fusion protein 1.2 D- Lemon scent 1.2.1 PCR LS 1.2.2 Ligation 1.2.3 pPS5 1.3 pPS3 and pPS4

LabWork

B- Construction of the fusion protein

We made a classic exttraction of plasmids from the liquid cultures.

Check by electrophoresis if it worked and send it for sequencing.

D- Lemon scent

by Mélanie

PCR LS

Electrophoresis of the PCR made Yesterday

[photo]

The PCR have success so I use the PCR clean up kit to purify it

[photo] Electrophoresis after purification

Ligation

We already have some pPSI digested and dephosphorelated

So I do a ligation:

component	volume
H2O	5μl
buffer	2μl
ligase	1μl
pPSI	2μl
LS PCR	10μl

2 hours at room temperature and over night at 4°

pPS5

Plasmid exctraction using the kit digestion by SalI

component	volume
H2O	11μl
buffer	5μl
SalI	2μl
pPS5	30μl

Electrophoresis

We saw that we have something strang with our plasmid but we wil check it tomorrow with a good ladder (the ladder here was normally on the well 1)

pPS3 and pPS4

We digest the plasmid by HindIII to direct our insert

component	volume
H2O	6μl
buffer	1μl
HindIII	1μl
pPS3/4	2μl

well 1 = ladder well 2-3-4 = pPS3 Well 5-6-7 = pPS4

results are very strange