Notebook

25.06.2013

Digest

Investigator: Dominik

Aim: Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with EcoRI and PstI

1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
0.3 µl EcoRI
0.3 µl PstI
1 µl CutSmart
7.4 µl ddH2O

The samples were incubated for 1 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

Worked as expected.

26.06.2013

Digest

Investigator: Dominik

Aim: Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9

1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)
0.5 µl EcoRI
0.5 µl SpeI
2 µl CutSmart
12 µl ddH2O

1 µl Plasmid (pSB1C3-iBB9)
0.5 µl XbaI
0.5 µl PstI
2 µl CutSmart
12 µl ddH2O

1 µl Plasmid (pSB1A3)
0.5 µl EcoRI
0.5 µl PstI
2 µl CutSmart
12 µl ddH2O

The samples were incubated for 1.5 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

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).

Ligation

Investigator: Dominik

Aim: Assemble the biobricks iBB10+iBB9, iBB11+iBB9, iBB12+iBB9 and iBB13+iBB9 into the vector pSB1A3.

2 µl vector DNA (pSB1A3)
2 µl insert 1 DNA (iBB9)
2 µl insert 2 DNA (iBB10, iBB11, iBB12 or iBB13)
2 µl 10x T4 DNA ligase buffer
2 µl T4 DNA ligase
2 µl ddH2O

The samples were incubated overnight at room temperature.

27.06.2013

Transformation

Investigator: Dominik

Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

Inoculation

Investigator: Dominik

Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.