Team:Marburg/Notebook:July

From 2013.igem.org

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<tr>
<tr>
<td>
<td>
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<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
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<img src="https://static.igem.org/mediawiki/2013/c/c5/Mr-Gel_2013-07-01_1.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
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<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl Ethidium bromide in 50 ml gel</li>
<li>10 µl Ethidium bromide in 50 ml gel</li>
-
<li>4 µl 2-log DNA Ladder</li>
+
<li>6 µl GeneRuler&trade; 2-log DNA Ladder (NEB)</li>
</ul>
</ul>
</p>
</p>
-
<p>
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<p>
-
<span class="exp">Expectations</span>
+
<table class="gel">
-
<ul class="exp">
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<colgroup>
-
<li>Lane 2: 479 + 2,581 + 6,000 bp</li>
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<col width="10%" />
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<li>Lane 3: 1,874 + 6,000 bp</li>
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<col width="2%" />
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<li>Lane 4: 2,160 + 3,650 + 6,000 bp</li>
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<col width="50%" />
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                                                        <li>Lane 5: 1,823 + 6,000 bp</li>
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<col width="5%" />
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                                                        <li>Lane 6: 1,199 + 2,682 + 6,000 bp</li>
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<col width="33%" />
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                                                        <li>Lane 7: 898 + 6,000 bp</li>
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</colgroup>
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                                                        <li>Lane 8: 693 + 6,000 bp</li>
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<thead>
-
</ul>
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<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Expectations</th>
 +
</thead>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR08 and iMR37</td>
 +
<th>&nbsp;</th>
 +
<td>479 bp</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR34 and iMR37</td>
 +
<th>&nbsp;</th>
 +
<td>1874 bp</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR12 and iMR37</td>
 +
<th>&nbsp;</th>
 +
<td>2160 bp + 3650 bp</td>
 +
</tr>
 +
                                                                <tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR31 and iMR38</td>
 +
<th>&nbsp;</th>
 +
<td>1823 bp</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR11 and iMR38</td>
 +
<th>&nbsp;</th>
 +
<td>1199 bp + 2682 bp</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR5 and iMR38</td>
 +
<th>&nbsp;</th>
 +
<td>898 bp</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<th>&nbsp;</th>
 +
<td>PCR with iMR9 and iMR38</td>
 +
<th>&nbsp;</th>
 +
<td>693 bp</td>
 +
</tr>
 +
</table>
</p>
</p>
-
<p>We received all expected fragments. Assembly of biobricks was successful.</p>
+
<p>We receive all expected fragments.</p>
</td>
</td>
-
</tr>
 
</tbody>
</tbody>
</table>
</table>
</div>
</div>
 +
<p>Assembly of biobricks was successful.</p>
</fieldset>
</fieldset>

Revision as of 20:51, 3 October 2013

Notebook: July

Anmerkung

Die Teile von Dominik müssen unbedingt noch überarbeitet werden!

01.07.2013

PCR
Investigator: Alex
Aim: Check correct assembly of the plasmid for antibody production
Volume Reagent   Temp (°C) Time
1 µl pSB1A3-iBB486476315   95 3 min
0.5 µl Primer forward   95 30 sec
0.5 µl Primer reverse   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
17 µl ddH2O

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl Ethidium bromide in 50 ml gel
  • 6 µl GeneRuler™ 2-log DNA Ladder (NEB)

Lane   Content   Expectations
2   PCR with iMR08 and iMR37   479 bp
3   PCR with iMR34 and iMR37   1874 bp
4   PCR with iMR12 and iMR37   2160 bp + 3650 bp
5   PCR with iMR31 and iMR38   1823 bp
6   PCR with iMR11 and iMR38   1199 bp + 2682 bp
7   PCR with iMR5 and iMR38   898 bp
8   PCR with iMR9 and iMR38   693 bp

We receive all expected fragments.

Assembly of biobricks was successful.

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

Expectations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.

15.07.2013

Sequencing
Investigator: Franzi
Aim: Construction of antibody-vector → Sequencing.

Sequencing pSB1A3 iBB486476315 K1 with primer 33 (iBB8 fw) AGBOOOK 275 and primer 34 (iBB8 rv) AGBOOOK 276.

16.07.2013

PCR
Investigator: Alex
Aim: Check ligation of iBB10/11/12/13 + iBB9 in pSB1A3
Volume Reagent   Temp (°C) Time
1 µl Template DNA   95 3 min
0.5 µl Primer forward   95 30 sec
0.5 µl Primer reverse   60 30 sec x60
0.5 µl dNTPs   72 1 min
0.5 µl Phusion polymerase   72 5 min
5 µl Phusion buffer 5x   4 Hold
17 µl ddH2O

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Expectations (upper gel)

  • Lane 2-4: 1,008 bp
  • Lane 5-7: 927 bp

Expectations (lower gel)

  • Lane 2-3: 903 bp
  • Lane 4-7: 888 bp

The upper gel showed the expected bands, whereas these bands were missing in the lower gel.
Sequencing
Investigator: Alex
Aim: Evaluate sequencing result of iBB8 in pSB1A3-iBB486476315
DNA / Biobrick Used primer Identities Gaps
BBa_K1071008 AB_plasmid_8 forward 941/941 (100%) 0
AB_plasmid_8 reverse 917/918 (100%) 1

Due to unreliabilities of the chain stop method by Sanger at the end of a DNA sequence, the detected gap can be considered as uncertain. This thesis is supported by 100% identity in the complementary strand.

The sequenced biobrick was believed to the accurate.

17.07.2013

Digest
Investigator: Alex
Aim: Digest of pSB1A3-iBB10/11/12/13+9 with XbaI and PstI, and pSB1C3-iBB4 with SpeI and PstI.

  • 2 µl Plasmide DNA
  • 0.6 µl Enzyme 1 (XbaI or SpeI, resp.)
  • 0.6 µl Enzyme 2 (PstI)
  • 2 µl CutSmart
  • 14.8 µl ddH2O
Ligation and transformation
Investigator: Alex
Aim: Assemble iBB4 in vectors pSB1A3-iBB10/11/12/13
  • Insert (iBB4) und plasmids were transformed using a 3:1 ratio.
  • Ligation samples were incubated ON at 16 °C.
  • Ligation products (pSB1A3-iBB4+10/11/12/13+9) were transformed into chemical competent DH5α cells (30 min ice, 90 sec 42°C, 45 min 37°C + 900 µl LB) and incubated ON at 37 °C.

18.07.2013

Inoculation
Investigator: Alex
Aim: Inoculation of colonies for midi plasmid preparation

Several clones of pSB1A3-iBB486476315/-iBB496315/-iBB395416 were picked and inoculated in 100 ml LB medium containing ampicillin.

Cultures were incubated ON at 37 °C.

19.07.2013

Midiprep
Investigator: Christian
Aim: Preparing the vectors for Phaeo → Midiprep of antibody-vector, PNR-test-vector and PfcpB-test-vector.

Midiprep of pSB1A3 iBB486476315, pSB1A3 iBB496315 and pSB1A3 iBB395416 (“QIAprep Spin Midiprep Kit” (Qiagen, Düsseldorf)).

22.07.2013

Digest
Investigator: Christian
Aim: Construction of signal peptide-vectors → Digestion of pSB1C3 iBB4, iBB10+9 and iBB13+9.
  • X µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 2 µl Cut Smart buffer
  • ad 20 µl H2O

Sample:

Digestion of 5 µl iBB4 with SpeI and PstI.

Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with XbaI and PstI.

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)
Ligation
Investigator: Christian
Aim: Construction of signal peptide-vectors → Ligation of iBB10+9 and iBB13+9 into pSB1C3 iBB4.
  • 1 µl vector (pSB1C3 iBB4)
  • either 5 µl iBB10+9
  • or 5 µl iBB13+9
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • 11 µl H2O

The samples were incubated for 2 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of signal peptide-vectors → Transformation of pSB1C3 iBB4-10-9 and pSB1C3 iBB4-13-9 into E. coli.

Transformation of the ligations into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LBCM, oN 37°C.

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

Expectations

  • 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

Expectations

  • 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

Expectations

  • 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

Expectations

  • 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

Expectations

  • 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.

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

Expectations

  • 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.

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

Expectations

  • 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.

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