Construction of pGGDestTol2LC - sgRNA vectors
Ligate the U6x : sgRNA cassettes into a pGGDestTol2LC vector by mixing the following components :
Incubate mixture at 37 °C for 20 min .
Incubate at 16 °C for 15min .
Incubate at 80 °C for 15min .
The reaction is ready for transformation ( use 5 µl of the ligation and plate 50 % of the transformants ) .
Transform and spread onto ampicillin ( 100 µg / ml ) plates .
