Team:Goettingen/protocol Plasmid Con

BP recombination reaction

Perform a BP recombination reaction between an attB-flanked DNA fragment and an attP-containing donor vector to generate an entry clone.
Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
     attB-PCR product    0.6 μl
     pDONR™ vector (supercoiled, 150 ng/μl)    0.2 μl
Vortex BP Clonase™ enzyme mix briefly.
Add 0.2 μl to the components above and mix well by vortexing briefly twice.
Immediately back into -80°C!
Incubate reaction at 25°C for 1 hour.
Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
Transform competent E. coli and select for the appropriate antibiotic-resistant entry clones.

LR recombination reaction

Perform an LR recombination reaction between an attL-containing entry clone and an attR-containing destination vector to generate an expression clone.
Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
     Entry clone (supercoiled, 100-300 ng)    0.6 μl
    Destination vector (supercoiled, 150 ng/μl)    0.2 μl
Vortex LR Clonase™ enzyme mix briefly. Add 0.2 μl to the components above and mix well by vortexing briefly twice.
Immediately back into -80°C!
Incubate reaction at 25°C for 1 hour.
Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
Transform competent E. coli and select for the appropriate antibiotic-resistant expression clones.