Team:Paris Saclay/Notebook/September/3


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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


Electrophoresis of the PCR made Yesterday

0309 PCR LS + extraction pPS5.jpg

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

0309 purif LS.jpg


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

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


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°


Plasmid exctraction using the kit (picture is here)

digestion by SalI

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


0309 digestion SalI pPS5.jpg

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

0309 digestion HindIII pPS3 4.jpg

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

results are very strange Back to the calendar