Team:Marburg/Notebook:March

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11.11.2011

Investigator: X,Y

Aim: We attempt to get results.

Wichtig ist, dass immer die verwendete Materialien (Plasmide, genomische DNA [aus welchen Stamm], Primer) genau benannt und/oder ggf. irgendwo nummeriert werden. Es sollte alles nachvollziehbar sein, wann ihr mit welchen Proben was gemacht habt.

Auf den folgenden Seiten sind Beispiele, wie wir uns das Notebook vom Aufbau und Detailgrad vorstellen.

17.04.2013

Ligation

Investigator: Franzi, Lucas

Aim: Assemble the Bricks 1, 3, 4, 5, 6, 7 and 8 into the vector pSB1C3.

5 µl vector DNA (pSB1C3)
20 µl insert DNA (1, 3, 4, 5, 6, 7 or 8)
3 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase

The samples were incubated for 14 h at 18° C.

Digest

Investigator: Christian, Patrick

Aim: Digest of pSB1C3-J04450 with MluI and HindIII.

4 µl DNA (pSB1C3-J04450)
1 µl MluI
1 µl HindIII
1,5 µl 10x red buffer
1,5 µl 10x orange g loading buffer
6 µl H2O

The samples were incubated for 4 h at 37° C.

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

We don’t receive all expected fragments. The expected fragment in lane 1 is missing.

18.04.2012

Transformation

Investigator: Franzi, Christian

Aim: Transformation of the plasmid DNA pB201-40JO in E. coli for amplification.

The chemo competent E. coli DH5a cells were transformed with pB201-40JO DNA and plated on dYT-Amp-plates.
The plates were incubated over night at 37° C .

PCR

Investigator: Patrick, Lucas

Aim: Receive PvuII point-mutation on colonies 3 and 5 of E. coli pB201-40JO.

Volume	Reagent	Temp (°C)	Time
10 µl	5x Buffer	95	3 min
1,5 µl	Primer fwd 13	95	3 sec
1,5 µl	Primer rev 14	58	30 sec	x17
1 µl	Template	72	1 min
36 µl	H2O	7 min	3 min
1 µl Phusion	Phusion Polymerase	4	Hold

Gel electrophoresis

Gel substances

1% Agarose gel
10 µl RedSafe in 50 ml gel
x µl Hyper Ladder

Expactations

Lane 1: 5300 kbp
Lane 2: 3300 kbp
Lane 3: 1300 kbp

We don’t receive all expected fragments. The expected fragment in lane 1 is missing.