lab 208

Main purpose

• Gel purification of HAO
• PCR
• USER reaction

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)

Per reaction:

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

Run reaction for 40 min on 37C and 30 mins on 25C. Made doubles and negative. One reaction is incubated with 50 uL of Top10 competent cells, the other with 100 uL of our competent cells. Only heat shock Top10 for 30 seconds, the other for 90 sec. Incubation for 2 h with 400 uL of SOC, afterwards plating on 30ug/mL kanamycin plates. 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.

Rehydration of primers

Received new primers. Rehydration with 150uL MilliQ to obtain stock solution and store in -80. 10uL of stock solution mixed with 90 uL of MilliQ to obtain -20 working solution.

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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