Team:Marburg/Notebook:June

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Notebook: June

17.06.2013

Digest
Investigator: Alex
Aim: Digest of pSB1A3_3+9 and pSB1A3_4+9 with SpeI and PstI, and corresponding inserts pSB1C3_5+4+1+6 and pSB1C3_6+3+1+5 with EcoRI and PstI.
  • 1 µl DNA
  • 0.3 µl Enzyme 1 (SpeI or EcoRI, resp.)
  • 0.3 µl Enzyme 2 (PstI)
  • 1 µl Cut Smart
  • 7.4 µl ddH2O

The samples were incubated for 1.5 h at 37° C.

Approaches were unsewed by means of gel electrophoresis, corresponding bands were picked under UV, DNA extraction from gel was performed using DNA Gel Extraction Kit (QIAGEN)

Gel electrophoresis
gel-electrophoresis-image

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.

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

24.06.2013

PCR
Investigator: Alex
Aim: Check right assembly of biobrick BBa_K1071005 in the plasmid for antibody production
Volume Reagent   Temp (°C) Time
1 µl pSB1A3_486476315   95 3 min
0.5 µl Primer fwd   95 30 sec
0.5 µl Primer rev   60 30 sec x30
0.5 µl dNTPs   72 42 sec
0.5 µl Phusion polymerase   72 5 min
5 µl Phusion buffer 5x   4 Hold
add 25 µL ddH2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide 50 ml gel
  • 4 µl 2-log DNA ladder

Expactations

  • Lane 1: ~ 6,000 bp for pSB1A3_486476315
  • Lane 2: 280 bp for BBa_K1071005

Each sample contained 2 bands, that means we got all expected bands and it could be assumed that the insertion of BBa_K1071005 was successful.

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-electrophoresis-image

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-electrophoresis-image

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.

02.07.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9)
  • 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-electrophoresis-image

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.

23.07.2013

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB4+iBB11+iBB9 and pSB1C3-iBB4+iBB12+iBB9)
  • 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-electrophoresis-image

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.

24.07.2013

Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1C3-iBB4+iBB10+iBB9 and 4 colonies of pSB1C3-iBB4+iBB13+iBB9 were inoculated in 5 ml LB medium containing chloramphenicol.

Digest
Investigator: Dominik
Aim: Digest of pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9, pSB1C3-iBB6315 and pSB1A3.

  • 1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
gel-electrophoresis-image

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 iBB4+iBB11+iBB96315 and iBB4+iBB12+iBB96315 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 5 µl insert 1 DNA (iBB6315)
  • 5 µl insert 2 DNA (iBB4+iBB11+iBB9, iBB4+iBB12+iBB9)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 4 µl ddH2O

The samples were incubated for 2h at 16 °C.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

25.07.2013

Sequencing
Investigator: Dominik
Aim: Complete sequencing of pSB1A3-iBB496315.

8 new sequencing samples were sent out.

Miniprep
Investigator: Dominik
Aim: preparation of the plasmids pSB1A3-iBB4+iBB10+iBB9 and pSB1A3-iBB4+iBB13+iBB9.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB4+iBB10+iBB9 and pSB1C3-iBB4+iBB13+iBB9)
  • 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-electrophoresis-image

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

Two samples of each plasmid preparation showed the expected fragments.
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB4+iBB11+iBB96315 and 4 colonies of pSB1A3-iBB4+iBB12+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.

Competent cells
Investigator: Dominik
Aim: Overnight culture for making new aliquots of competent cells.

For making new aliquots of E. coli DH5α cells 50 ml LB medium were inoculated with 500 µl of an E. coli DH5α culture.

26.07.2013

Digest
Investigator: Dominik
Aim: Digest of pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9, pSB1C3-iBB6315 and pSB1A3.

  • 1000 ng Plasmid (pSB1C3-iBB4+iBB10+iBB9, pSB1C3-iBB4+iBB13+iBB9)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
gel-electrophoresis-image

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 iBB4+iBB10+iBB9, iBB4+iBB13+iBB9 and iBB96315 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 5 µl insert 1 DNA (iBB96315)
  • 5 µl insert 2 DNA (iBB4+iBB10+iBB9,iBB4+iBB13+iBB9)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 4 µl ddH2O

The samples were incubated for 1h at 16 °C.

Resulting in the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

Miniprep
Investigator: Dominik
Aim: preparation of the pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of ppSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 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-electrophoresis-image

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

Most of the preparations resulted in the expected fragments, all others were discarded.
Competent cells
Investigator: Dominik
Aim: New aliquots of competent cells (E. coli DH5α).

About 80 new aliquots were made.

02.07.2013

Sequencing
Investigator: Dominik
Aim: Analysis of the sequence of pSB1A3-iBB496315.

Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

30.07.2013

Digest
Investigator: Dominik
Aim: Digest of pSB1A3-iBB49 and pSB1C3-iBB6315.

  • 1000 ng Plasmid (pSB1A3-iBB49)
  • 0.5 µl PstI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
gel-electrophoresis-image

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).
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB4+iBB10+iBB96315 and the only single colony of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol. Additionally the remaining ligation preparation was transformed in E. coli DH5α?????.

31.07.2013

Ligation
Investigator: Dominik
Aim: Ligation of pSB1A3-iBB49 and iBB6315.

  • 2 µl vector DNA (pSB1A3-iBB49) (40 ng)
  • 5 µl insert DNA (iBB6315) (150 ng)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 9 µl ddH2O

The samples were incubated for 1h at 16 °C.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB49+iBB6315.

The chemo competent E. coli DH5α cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

Miniprep
Investigator: Dominik
Aim: Preparation of the pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The culture of pSB1A3-iBB4+iBB13+iBB96315 had a pinkish color and therefore was discarded. The plasmid pSB1A3-iBB4+iBB10+iBB96315 was isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB10+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB4+iBB10+iBB96315)
  • 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-electrophoresis-image

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.
Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

8 samples were sent out for sequencing.

Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

Both colonies of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing ampicillin.

01.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

2 additional sequence samples were sent out for sequencing.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB13+iBB96315 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-electrophoresis-image

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

After that, a gel electrophoresis was made, but failed and has to be repeated the next day
Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

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

02.07.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmid pSB1A3-iBB496315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)
  • 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-electrophoresis-image

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.

14.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

  • pSB1A3-iBB496315: Okay
  • pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp → Leu. Will be ignored
  • pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.

All plasmids are brought to Marian who will transform them in P. tricornutum cells.

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