lab ...

Main purpose

Who was in the lab

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.

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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