Team:Marburg/Notebook:August

From 2013.igem.org

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<a name="Anmerkung">Anmerkung</a>
 
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<p>Die Teile von Dominik müssen unbedingt noch überarbeitet werden!</p>
 
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<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
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<li>0.3 µl EcoRI</li>
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<li>0.3 µl <i>Eco</i>RI</li>
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<li>0.3 µl PstI</li>
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<li>0.3 µl <i>Pst</I></li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
<li>7.4 µl ddH2O</li>
<li>7.4 µl ddH2O</li>
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<p>The samples were incubated for 1 h at 37° C.</p>
<p>The samples were incubated for 1 h at 37° C.</p>
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<p>After that, a gel electrophoresis was made, but failed and has to be repeated the next day</p>
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<th colspan="2" class="title">Gel electrophoresis</th>
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<li>x µl Hyper Ladder</li>
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<span class="exp">Expectations</span>
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<li>Lane 1: 5300 kbp</li>
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<li>Lane 2: 3300 kbp</li>
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<li>Lane 3: 1300 kbp</li>
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Revision as of 19:31, 3 October 2013

Notebook: August

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 Pst
  • 1 µl CutSmart
  • 7.4 µl ddH2O

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

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.

02.08.2013

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.

03.08.2013

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

Expectations

  • 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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