TFKE39 Seminar : Project Course

1. TFKE39Seminar : Project Course Application of Molecular Biology ”tools” for cloning of a foreign gene David Grossekathöfer Antoine Malabirade Elodie Person Estelle Picq AdrienSoula Hélène Trottin Bo Kyung Kim 10/13/2011

2. 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 At the second PCR was not enough water added to the comercial sample The vector was restricted two times and purified three times 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.

3. PCR-Cycles Denaturation Annealing Elongation What we had to choose: the annealing temperature First PCR (did not work): • Annealing T = 66˚C • Taq DNA-Polymerase: Commercial Second PCR (a success!) • Annealing T = 60˚C • Taq DNA-Polymerase: Commercial + Home-made pET-28 (double restriction with NcoI and Hind III) T-tailed vector (without restriction)

4. 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)

5. First PCR results No PCR product Vector 110919

6. Second PCR results Home made Taq-polymerase Commercial Taq-polymerase 110922

7. Final results Our 4 samples PCR product of a tranformated colony 110926