Team:Marburg/Notebook:July

From 2013.igem.org

(Difference between revisions)
Line 82: Line 82:
                         <tr>
                         <tr>
                                 <td>17 µl</td>
                                 <td>17 µl</td>
-
                                 <td>ddH2O</td>
+
                                 <td>ddbidest. H2O</td>
                         </tr>
                         </tr>
</table>
</table>
Line 224: Line 224:
<li>0.3 µl PstI</li>
<li>0.3 µl PstI</li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 402: Line 402:
                         <tr>
                         <tr>
                                 <td>17 µl</td>
                                 <td>17 µl</td>
-
                                 <td>ddH2O</td>
+
                                 <td>ddbidest. H2O</td>
                         </tr>
                         </tr>
</table>
</table>
Line 545: Line 545:
                         </table>
                         </table>
                         <p>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.</p>
                         <p>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.</p>
-
<p>The sequenced biobrick was believed to the accurate.</p>  
+
<p>The sequenced biobrick is thought to the accurate.</p>  
</div>
</div>
</fieldset>
</fieldset>
Line 571: Line 571:
<li>0.6 µl Enzyme 2 (PstI)</li>
<li>0.6 µl Enzyme 2 (PstI)</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>14.8 µl ddH2O</li>
+
<li>14.8 µl ddbidest. H2O</li>
</ul>
</ul>
</div>
</div>
Line 590: Line 590:
<li>Insert (iBB4) und plasmids were transformed using a 3:1 ratio.</li>
<li>Insert (iBB4) und plasmids were transformed using a 3:1 ratio.</li>
<li>Ligation samples were incubated ON at 16 °C.</li>
<li>Ligation samples were incubated ON at 16 °C.</li>
-
                         <li>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.</li>
+
                         <li>Ligation products (pSB1A3-iBB4+10/11/12/13+9) were transformed into chemical competent DH5α cells (30 min on ice, 90 sec 42 °C, 45 min 37 °C + 900 µl LB) and incubated ON at 37 °C.</li>
</ul>
</ul>
</div>
</div>
Line 665: Line 665:
<li>1 µl enzyme 2</li>
<li>1 µl enzyme 2</li>
<li>2 µl Cut Smart buffer</li>
<li>2 µl Cut Smart buffer</li>
-
<li>ad 20 µl H2O</li>
+
<li>ad 20 µl bidest. H2O</li>
</ul>
</ul>
<p>Sample:</p>
<p>Sample:</p>
<p>Digestion of 5 µl iBB4 with <i>Spe</i>I and <i>Pst</i>I.</p>
<p>Digestion of 5 µl iBB4 with <i>Spe</i>I and <i>Pst</i>I.</p>
<p>Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with <i>Xba</i>I and <i>Pst</i>I.</p>
<p>Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with <i>Xba</i>I and <i>Pst</i>I.</p>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
</div>
</div>
Line 693: Line 693:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>1 µl T4 DNA ligase</li>
<li>1 µl T4 DNA ligase</li>
-
<li>11 µl H2O</li>
+
<li>11 µl bidest. H2O</li>
</ul>
</ul>
<p>The samples were incubated for 2 h at RT.</p>
<p>The samples were incubated for 2 h at RT.</p>
Line 711: Line 711:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of the ligations into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LB<sub>CM</sub>, oN 37°C.</p>
+
<p>Transformation of the ligations into <i>E. coli</i> DH5&alpha; (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LB<sub>CM</sub>, overnight at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 739: Line 739:
<li>0.3 µl PstI</li>
<li>0.3 µl PstI</li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 845: Line 845:
<li>0.5 µl SpeI</li>
<li>0.5 µl SpeI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
Line 853: Line 853:
<li>0.5 µl PstI</li>
<li>0.5 µl PstI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
Line 861: Line 861:
<li>0.5 µl PstI</li>
<li>0.5 µl PstI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 936: Line 936:
</p>
</p>
-
<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).</p>
+
<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).</p>
</td>
</td>
</tr>
</tr>
Line 961: Line 961:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl T4 DNA ligase</li>
<li>2 µl T4 DNA ligase</li>
-
<li>4 µl ddH2O</li>
+
<li>4 µl ddbidest. H2O</li>
</ul>
</ul>
<p>The samples were incubated for 2h at 16 °C.</p>
<p>The samples were incubated for 2h at 16 °C.</p>
Line 979: Line 979:
<div class="exp-content">
<div class="exp-content">
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.<br />
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.<br />
-
The plates were incubated over night at 37° C.</p>
+
The plates were incubated over night at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,035: Line 1,035:
<li>0.3 µl PstI</li>
<li>0.3 µl PstI</li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
</div>
</div>
Line 1,065: Line 1,065:
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Overnight culture for making new aliquots of competent cells.</span>
+
<span class="aim-desc">overnight at culture for making new aliquots of competent cells.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
Line 1,094: Line 1,094:
<li>0.5 µl SpeI</li>
<li>0.5 µl SpeI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
Line 1,102: Line 1,102:
<li>0.5 µl PstI</li>
<li>0.5 µl PstI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
Line 1,110: Line 1,110:
<li>0.5 µl PstI</li>
<li>0.5 µl PstI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,184: Line 1,184:
</table>
</table>
</p>
</p>
-
<p>Two expected fragments are present. The relevant fragments are cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).</p>
+
<p>Two expected fragments are present. The relevant fragments are cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).</p>
</td>
</td>
</tr>
</tr>
Line 1,209: Line 1,209:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl T4 DNA ligase</li>
<li>2 µl T4 DNA ligase</li>
-
<li>4 µl ddH2O</li>
+
<li>4 µl ddbidest. H2O</li>
</ul>
</ul>
<p>The samples were incubated for 1h at 16 °C.</p>
<p>The samples were incubated for 1h at 16 °C.</p>
Line 1,247: Line 1,247:
<li>0.3 µl PstI</li>
<li>0.3 µl PstI</li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 1,366: Line 1,366:
<li>0.5 µl SpeI</li>
<li>0.5 µl SpeI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
Line 1,374: Line 1,374:
<li>0.5 µl PstI</li>
<li>0.5 µl PstI</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
-
<li>ad 20 µl ddH2O</li>
+
<li>ad 20 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
</div>
</div>
Line 1,419: Line 1,419:
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl 10x T4 DNA ligase buffer</li>
<li>2 µl T4 DNA ligase</li>
<li>2 µl T4 DNA ligase</li>
-
<li>9 µl ddH2O</li>
+
<li>9 µl ddbidest. H2O</li>
</ul>
</ul>
<p>The samples were incubated for 1h at 16 °C.</p>
<p>The samples were incubated for 1h at 16 °C.</p>
Line 1,437: Line 1,437:
<div class="exp-content">
<div class="exp-content">
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates.<br />
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates.<br />
-
The plates were incubated over night at 37° C.</p>
+
The plates were incubated over night at 37 °C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 1,472: Line 1,472:
<li>0.3 µl PstI</li>
<li>0.3 µl PstI</li>
<li>1 µl CutSmart</li>
<li>1 µl CutSmart</li>
-
<li>7.4 µl ddH2O</li>
+
<li>7.4 µl ddbidest. H2O</li>
</ul>
</ul>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>

Revision as of 13:27, 4 October 2013

Notebook: July

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 ddbidest. H2O

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 is 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 ddbidest. H2O

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

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   pSB1A3-iBB10+9   5300 bp
3   pSB1A3-iBB11+9   3300 bp
4   pSB1A3-iBB12+9   1300 bp

Worked as expected.

3 out of 6 samples positive.

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 ddbidest. H2O

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)

Upper gel

Lane   Content   Expectations
2-4   pSB1A3-iBB10+9   1008 bp
5-7   pSB1A3-iBB11+9   927 bp

Lower gel

Lane   Content   Expectations
2-3   pSB1A3-iBB12+9   903 bp
4-7   pSB1A3-iBB13+9   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 is thought 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 ddbidest. H2O
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 on 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 bidest. 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.

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 bidest. 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 on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 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 ddbidest. H2O

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

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
2-5   pSB1C3-iBB4+11+9   1349 bp
6-8   pSB1C3-iBB4+12+9   1325 bp

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 ddbidest. H2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddbidest. H2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddbidest. H2O

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

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   pSB1C3-iBB4+11+9   1349 bp
3   pSB1C3-iBB4+12+9   1325 bp
4   pSB1C3-iBB6315   1082 bp
5   pSB1A3   2000 bp

All expected fragments were present. The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).
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 ddbidest. H2O

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 ddbidest. H2O

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

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 at 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 ddbidest. H2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddbidest. H2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddbidest. H2O

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

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   pSB1C3-iBB4+10+9   1430 bp
3   pSB1C3-iBB4+13+9   1310 bp
4   pSB1C3-iBB6315   1082 bp
5   pSB1A3   2000 bp

Two expected fragments are present. The relevant fragments are cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen).
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 ddbidest. H2O

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 ddbidest. H2O

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

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   pSB1A3-iBB4+10+96315   2392 bp
3   pSB1A3-iBB4+12+96315   2287 bp
4   pSB1C3-iBB39   913 bp
5   pSB1A3-iBB49   1142 bp

An expected double band is shown in Sample 1 (Vector + Insert). The other sample do not exhibit the expected band and are therefore rejected.
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 ddbidest. H2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddbidest. H2O

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

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 ddbidest. H2O

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 ddbidest. H2O

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

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
2-5   pSB1A3-iBB4+10+96315   2392 bp

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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