Team:Marburg/Notebook:March

From 2013.igem.org

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<p>Digestion of pPha-NR with <i>EcoR</i>I and <i>Nco</i>I.</p>
<p>Digestion of pPha-NR with <i>EcoR</i>I and <i>Nco</i>I.</p>
<p>Digestion of pPha-T1 with <i>EcoR</i>I and <i>Nde</i>I.</p>
<p>Digestion of pPha-T1 with <i>EcoR</i>I and <i>Nde</i>I.</p>
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<p>The samples were incubated for 1 h at 37° C.</p>
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<p>The samples were incubated for 1 h at 37 °C.</p>
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<td>1 µl</td>
<td>1 µl</td>
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<td>Primer fw (100pmol/µl)</td>
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<td>Primer fw (100 pmol/µl)</td>
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<td>1 µl</td>
<td>1 µl</td>
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<p>Digestion of iBB3 (P<sub>fcpB</sub>) with <i>Pvu</i>II.</p>
<p>Digestion of iBB3 (P<sub>fcpB</sub>) with <i>Pvu</i>II.</p>
<p>Digestion of IBB4 (P<sub>NR</sub>) with <i>Pst</i>I.</p>
<p>Digestion of IBB4 (P<sub>NR</sub>) with <i>Pst</i>I.</p>
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<p>The samples were incubated for 1 h at 37° C.</p>
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<p>The samples were incubated for 1 h at 37 °C.</p>
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Revision as of 12:54, 4 October 2013

Notebook: March

20.03.2013

Transformation
Investigator: Christian
Aim: Preparation of BioBrick templates → Transformation into E. coli DH5α.

Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with bidest. H2O) in E. coli DH5α (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

21.03.2013

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation of transformants for miniprep.

2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LBamp, overnight at 37°C.

pCAT is a cosmid, transformation failed.

22.03.2013

Miniprep
Investigator: Christian
Aim: Preparation of BioBrick templates → Miniprep of the template-plasmids.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Preparation of BioBrick templates → Control digestion of the template plasmids.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 4 µl buffer 2
  • 31 µl bidest. H2O

Samples:

Digestion of pPha-NR with EcoRI and NcoI.

Digestion of pPha-T1 with EcoRI and NdeI.

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   pPha-NR   2868 bp + 992 bp
4+5   pPha-T1   3554 bp + 541 bp

All positive.
PCR
Investigator: Christian
Aim: Construction of BioBricks → Deletion of restriction sites in iBB3, iBB4 and iBB5 via mutagenesis PCR.

Mutagenesis PCR of PfcpB (iBB3), PNR (iBB4), and TfcpA (iBB5).

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw (100 pmol/µl)   95 30 sec
1 µl Primer rv (100 pmol/µl)   65 45 sec x19
1 µl Template pPha-NR (diluted 1:100)   72 4:10 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
35 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content    
2   PfcpB    
3   PNR    
4   PfcpA    
5   PfcpB    
6   PNR    
7   PfcpA    

Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion of the template plasmid.

Digestion of the template with 1 µl DppI.

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of mutagenised plasmids.

Transformation of 1 µl PCR-product into E. coli DH5α (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

26.03.2013

Inoculation
Investigator: Christian
Aim: Construction of BioBricks → Inoculation of transformants for miniprep.

Transformation of iBB5 failed, new mutagenesis primers ordered.

3 colonies of iBB3 and iBB4 inoculated in 5 ml LBamp, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

Inoculation of Ca-competent E. coli DH5α in 2x5 ml LB, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation for glycerol stocks.

PPha-NR and pPha-T1 were inoculated in 5 ml LB, overnight at 37°C (for glycerol stocks).

27.03.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks → Miniprep of transformants.

Miniprep of of iBB3 and iBB4 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

3 ml E. coli DH5α pre-culture inoculated in 100 ml LB.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Amplification of iBB2 via PCR.

PCR on CAT-cassette (iBB2):

Volume Reagent   Temp (°C) Time
1 µl Q5 Polymerase   95 3 min
1 µl Primer fw (1pmol/µl)   95 30 sec
1 µl Primer rv (1pmol/µl)   65 30 sec x26
1 µl Template   72 1 min
1 µl dNTPs   72 7 min
10 µl 5x Q5-buffer   4 Hold
36 µl bidest. H2O  

The PCR was cleaned via 1% agarose gel: Relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Transformation
Investigator: Christian
Aim: Production of chemo-competent E. coli → Control of cells.

Competent cells are harvested and frozen.

Controlled with several Transformations:

  • on LB
  • on LBamp
  • on LBamp with 1 µl pPha-T1
  • cells from AG Bange as control

30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, overnight at 37°C.

Digest
Investigator: Christian
Aim: Construction of BioBricks → Control digestion of mutations.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 1 µl buffer
  • 5 µl bidest. H2O

Samples:

Digestion of iBB3 (PfcpB) with PvuII.

Digestion of IBB4 (PNR) with PstI.

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
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Comment
2   PfcpB 1   no cut, vector in different forms
3   PfcpB 2  
4   PfcpB 3  
5   PNR 1   1 cut → linearized plasmid
6   PNR 2  
7   PNR 3  

28.03.2013

Sequencing
Investigator: Christian
Aim: Construction of BioBricks → Sequencing.

IBB3 and iBB4 with point mutations prepared for sequencing:

  • AGBOOOK 128: iBB4 1
  • AGBOOOK 129: iBB4 2
  • AGBOOOK 130: iBB4 3
  • AGBOOOK 131: iBB3 1
  • AGBOOOK 132: iBB3 2
  • AGBOOOK 133: iBB3 3

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