lab ...

Main purpose

Who was in the lab

Kristian, Natalia, Julia, Henrike

Procedure

gel purification

Gel purified HAO fragment for USER cloning and pZA21 with endings for cloning with the 2-part Nir. Used QIAEX kit (without columns).

PCR

Set up PCR to amplify AMO as fragment for USER cloning. Run triplicates on 50C and 3:00 and dublicates on a ramp program 56C -> 50C with 0.1C/s and 3:00

USER reaction with HAO and pZA21 (native)

Procedure as protocolled.

Per reaction:

• USER enzyme - 1 uL
• NEB buffer 4 - 0.5 uL
• 10x BSA - 0.5 uL
• backbone - 1 uL
• fragment - 7 uL

Made doubles and negative. One reaction is incubated with top10 competent cells, the other with our competent cells. Cells will be grown on the bench instead of in the incubator.

LB+Kan plates

Made agar plates from LB medium and added Kanamycin to final concentration of 30 ug/ml.

Results

Gels

1% gel for PCR of cytochromes with His-tag

• neg
• cycAX with His-tag at 45C
• cycAX with His-tag at 45C
• cycAX with His-tag at 50C
• cycAX with His-tag at 50C
• 1 kb ladder

1% gel for PCR products of Nir

• 1 kb ladder
• Nir part 2
• Nir part 2
• Nir part 1
• Nir part 1
• neg

Conclusion

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