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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