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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 1.3 Digestion 1.3.1 Ligation 1.3.2 pPS5 1.4 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

well 2-3 = PCR with Dream taq or Vent taq

(well 4-5 = checking of the pps5 Extraction)

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

Electrophoresis after purification

Digestion

In order to clone LS in pPSI, i Digest it by PacI

component	volume
H2O	3μl
buffer	1μl
PacI	1μl
PCR purify	5μl

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 (picture is here)

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