Team:Marburg/Notebook:Template

From 2013.igem.org

(Difference between revisions)
Line 75: Line 75:
<ul class="digest">
<ul class="digest">
<li>4 µl DNA (pSB1C3-J04450)</li>
<li>4 µl DNA (pSB1C3-J04450)</li>
-
<li>1 µl MluI</li>
+
<li>1 µl <i>Mlu</i>I</li>
-
<li>1 µl HindIII</li>
+
<li>1 µl <i>Hind</i>III</li>
<li>1,5 µl 10x red buffer</li>
<li>1,5 µl 10x red buffer</li>
<li>1,5 µl 10x orange g loading buffer</li>
<li>1,5 µl 10x orange g loading buffer</li>
Line 103: Line 103:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
</ul>
</ul>
</p>
</p>
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<p><ul class="digest">
<p><ul class="digest">
<li>1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)</li>
<li>1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)</li>
-
<li>0.5 µl EcoRI</li>
+
<li>0.5 µl <i>EcoR</i>I</li>
-
<li>0.5 µl SpeI</li>
+
<li>0.5 µl <i>Spe</i>I</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
<li>ad 20 µl ddH2O</li>
<li>ad 20 µl ddH2O</li>
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<p><ul class="digest">
<p><ul class="digest">
<li>1000 ng Plasmid (pSB1C3-iBB6315)</li>
<li>1000 ng Plasmid (pSB1C3-iBB6315)</li>
-
<li>0.5 µl XbaI</li>
+
<li>0.5 µl <i>Xba</i>I</li>
-
<li>0.5 µl PstI</li>
+
<li>0.5 µl <i>Pst</i>I</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
<li>ad 20 µl ddH2O</li>
<li>ad 20 µl ddH2O</li>
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<p><ul class="digest">
<p><ul class="digest">
<li>1000 ng Plasmid (pSB1A3)</li>
<li>1000 ng Plasmid (pSB1A3)</li>
-
<li>0.5 µl EcoRI</li>
+
<li>0.5 µl <i>EcoR</i>I</li>
-
<li>0.5 µl PstI</li>
+
<li>0.5 µl <i>Pst</i>I</li>
<li>2 µl CutSmart</li>
<li>2 µl CutSmart</li>
<li>ad 20 µl ddH2O</li>
<li>ad 20 µl ddH2O</li>
Line 320: Line 320:
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
<li>10 µl RedSafe in 50 ml gel</li>
-
<li>x µl Hyper Ladder</li>
+
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
</ul>
</ul>
</p>
</p>

Revision as of 19:43, 30 September 2013

Notebook

11.11.2011

Überschrift
Investigator: X,Y
Aim: We attempt to get results.

Wichtig ist, dass immer die verwendete Materialien (Plasmide, genomische DNA [aus welchen Stamm], Primer) genau benannt und/oder ggf. irgendwo nummeriert werden. Es sollte alles nachvollziehbar sein, wann ihr mit welchen Proben was gemacht habt.

Auf den folgenden Seiten sind Beispiele, wie wir uns das Notebook vom Aufbau und Detailgrad vorstellen.

17.04.2013

Ligation
Investigator: Franzi, Lucas
Aim: Assemble the Bricks 1, 3, 4, 5, 6, 7 and 8 into the vector pSB1C3.
  • 5 µl vector DNA (pSB1C3)
  • 20 µl insert DNA (1, 3, 4, 5, 6, 7 or 8)
  • 3 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase

The samples were incubated for 14 h at 18° C.

Digest
Investigator: Christian, Patrick
Aim: Digest of pSB1C3-J04450 with MluI and HindIII.
  • 4 µl DNA (pSB1C3-J04450)
  • 1 µl MluI
  • 1 µl HindIII
  • 1,5 µl 10x red buffer
  • 1,5 µl 10x orange g loading buffer
  • 6 µl H2O

The samples were incubated for 4 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)

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.

18.04.2012

Transformation
Investigator: Franzi, Christian
Aim: Transformation of the plasmid DNA pB201-40JO in E. coli for amplification.

The chemo competent E. coli DH5a cells were transformed with pB201-40JO DNA and plated on dYT-Amp-plates.
The plates were incubated over night at 37° C .

PCR
Investigator: Patrick, Lucas
Aim: Receive PvuII point-mutation on colonies 3 and 5 of E. coli pB201-40JO.
Volume Reagent   Temp (°C) Time
10 µl 5x Buffer   95 3 min
1,5 µl Primer fwd 13   95 3 sec
1,5 µl Primer rev 14   58 30 sec x17
1 µl Template   72 1 min
36 µl H2O   7 min 3 min
1 µl Phusion Phusion Polymerase   4 Hold

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.
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
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)

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-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

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

8 new sequencing samples were sent out.

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.

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.

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