Difference between revisions of "Template:Team:TU Eindhoven/Timeline HTML"

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<li><span class="activiteit">Plasmid isolation, followed by sequencing of the insert on MCS1 </span></li>
 
<li><span class="activiteit">Plasmid isolation, followed by sequencing of the insert on MCS1 </span></li>
 
</ul>
 
</ul>
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<br \>
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<span class="activiteit"> Protein expression:</span>
 +
<ul class="activiteitlijst">
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<li><span class="activiteit">Double transformation of pET-Duet-1 (MCS1) and pEVOL in BL21. </span></li>
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<li><span class="activiteit">Culturing & making a glycerol stock </span></li>
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</ul>
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<br \>
 
<br \>
 
<br \>
 
<span class="activiteit"> Traditional cloning: <br />The sequencing results are positive, everything is built in correctly.</span>
 
<span class="activiteit"> Traditional cloning: <br />The sequencing results are positive, everything is built in correctly.</span>

Revision as of 10:42, 17 July 2015





Timeline



Week 26

- Preparing for take-off


  • Inventory of the lab supplies
  • Pouring LB Agar plates
  • Amplification of the pET-Duet-1 vector
  • Assessment of the safety requirements
  • Preparing stocks for antibiotics, glycerol, LB & MilliQ


Week 27

- The Clone Wars


Gibson Assembly:
  • Linearizing pET-Duet-1 for Gibson Assembly
  • Our first Gibson Assembly!

Traditional cloning:
  • Amplification of the pET-Duet-1 vector
  • Nde1 & Kpn1 digestion of the pET-Duet-1 vector (MCS-2) for traditional cloning


Week 28

- Et tu, pET-Duet?


Gibson Assembly:
  • Linearizing pET-Duet-1 for Gibson Assembly and debugging
  • Digestion of the template using DpnI
  • Running a gel to check whether the linearization was successful
  • Gibson Assembly for MCS1
  • Transformation and amplification of the plasmid

Traditional cloning:
  • Amplification of the inserts
  • Xbal & Pstl digestion of the pET-Duet-1 vector (MCS-1)
  • Xba1 & Pst1 digestion of the inserts (MCS-1)
  • Nde1 & Kpn1 digestion of the inserts (MCS-2)
  • Ligation
  • Transformation in NB
  • Colony PCR & gel electrophorese: The inserts are succesfully ligated
  • Culturing of the colonies with the correct plasmid
  • Making a glycerol stock & sending the DNA for sequencing


Week 29

- Hopeful results


Gibson Assembly:
  • Plasmid isolation, followed by sequencing of the insert on MCS1

Protein expression:
  • Double transformation of pET-Duet-1 (MCS1) and pEVOL in BL21.
  • Culturing & making a glycerol stock


Traditional cloning:
The sequencing results are positive, everything is built in correctly.