Difference between revisions of "Template:Team:TU Eindhoven/Timeline HTML"
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<li><span class="activiteit">Double transformation, culturing and protein expression of pET-Duet-1 (MCS-1 & MCS-2) </span></li> | <li><span class="activiteit">Double transformation, culturing and protein expression of pET-Duet-1 (MCS-1 & MCS-2) </span></li> | ||
<li><span class="activiteit">The click reaction occured according to FACS results </span></li> | <li><span class="activiteit">The click reaction occured according to FACS results </span></li> | ||
| + | </ul> | ||
| + | <br \> | ||
| + | <span class="activiteit"> Traditional cloning</span> | ||
| + | <ul class="activiteitlijst"> | ||
| + | <li><span class="activiteit">The vector which already contained MCS-1 is digested at MCS-2 </span></li> | ||
| + | <li><span class="activiteit">Ligation of vector & insert. This results in a plasmid containing MCS-1 (OmpX-NanoLuc) and MCS-2 (OmpX-neongreen) </span></li> | ||
</ul> | </ul> | ||
Revision as of 07:42, 27 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
- Linearization of the vector on MCS2
- Gibson Assembly of the second MCS
- Colony PCR of MCS2, showing promising results!
- Culturing and preparing for protein expression
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.
Week 30
- The moment of truth
Gibson Assembly:
- Plasmid Isolation, followed by sequencing of the insert on MCS2
Protein expression of the plasmids containing MCS-1:
- Culturing and protein expression of pET-Duet-1 (MCS-1)
- Labelling the bacteria with DBCO-PEG4-Tamra
- FACS results don't show the click reaction...
Double transformation & protein expression of the plasmids containing MCS-1 & MCS-2:
- Double transformation, culturing and protein expression of pET-Duet-1 (MCS-1 & MCS-2)
- The click reaction occured according to FACS results
Traditional cloning
- The vector which already contained MCS-1 is digested at MCS-2
- Ligation of vector & insert. This results in a plasmid containing MCS-1 (OmpX-NanoLuc) and MCS-2 (OmpX-neongreen)