208 lab

Main purpose today

Make gel purifications to enable USER-reactions and to make PCR of the fragments missing.

Who was in the lab

Kristian

Procedure

Purification gel 1,2 and 3

• 1kb ladder
• Nir
• Nir
• Nir
• Nir
• Nir
• Nir
• Nir
• Nir
• HAO

• 1kb ladder, NEB
• cycAX
• cycAX
• cycAX
• cycAX
• AMO
• AMO
• AMO
• AMO
• HAO
• HAO
• HAO

• 100 bp ladder, NEB
• RFP
• RFP
• RFP

PCR of AMO

Did PCR on AMO on two programs: 1-3 on 54°C 4-6 on 52°C Both with the annealing time 3 min.

USER-reactions

There was made USER-reactions with all fragments after purification. AMO was purified even though it was only one very faint band; the others were bright band. The following plates where made:

• RFP USER - RFP inside pZA21
• AMO USER - AMO inside pZA21
• HAO USER - HAO inside pZA21
• cycAX USER - cycAX inside pZA21
• Nir USER - Nir inside pZA21
• Neg. USER - pZA21 linearized only

Conclusion

PCR of AMO with USER-primers was not a success we will see if the new PCR have done better. We will evaluate the transformants on Monday with colony-PCR.