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Revision as of 14:09, 15 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
Traditional cloning:
The sequencing results are positive, everything is built in correctly.