Transcript Document

TFKE39
Seminar : Project Course
Application of Molecular Biology ”tools”
for cloning of a foreign gene
David Grossekathöfer
Antoine Malabirade
Elodie Person
Estelle Picq
Adrien Soula
Hélène Trottin
Bo Kyung Kim
10/13/2011
Schedule
First PCR attempt : Too high dNTP concentration
Why? we added:
- the buffer that already contained dNTP
- Extra dNTP
Consequence: dNTP is absorbing Mg2+ which is
important for Taq Polymerase activity
 Taq Polymerase inactive and PCR failed
PCR to
amplify our
gene
With the
home-made
polymerase
With the
commercial
polymerase
Purification
Purification
Ligation with Tvector
Restriction +
Purification
In contrast to a few other groups
we did not used alkaline
phosphatase to dephosphorylise
the 5’-Ends at our vector. If you
use two diverse restriction
enzymes this is not necessary.
At the second PCR was
not enough water added
to the comercial sample
Vector
The vector was
restricted two times
and purified three
times
Restriction
Purification
Ligation with
PET vector
Transformation
in E. coli
Picking 4
colonies for
PCR
Transformation
in E. coli
END (no
colonies)
2
PCR-Cycles
Denaturation
Annealing
Elongation
What we had to choose: the annealing temperature
First PCR (did not work):
Second PCR (a success!)
Annealing T = 66˚C
 Taq DNA-Polymerase:
Commercial


Annealing T = 60˚C
 Taq DNA-Polymerase:
Commercial + Home-made
pET-28 (double
restriction with
NcoI and Hind III)
T-tailed vector
(without
restriction)
3
Control transformation
For our control we added the vector but not
the insert.
Unexpectedly there were colonies on our
Agarplate (relative high concentration: 30 %,
uniform look)
 Self-ligation because of differently working
restriction enyzmes (Antibiotic resistance is
located in the vector)
4
First PCR results
No PCR
product
Vector
110919
5
Second PCR results
Commercial Taq- Home made
Taq-polymerase
polymerase
110922
6
Final results
Our 4 samples
PCR product of a
tranformated colony
110926
7