Notebook: March

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