CLONING FROM LOW MELT AGAROSE

competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl₂ transformation. Place 100 µl of TB buffer in a tube on ice. Melt the ligation mix at 65℃ for 5 minutes and add a 5-15 µl aliquot of the ligation mix to the TB solution and vortex quickly. Add 200 µl of competent E. coli to the tube, vortex lightly. Incubate 30-60 minutes on ice, heat shock 90 seconds at 42℃, and return to ice 2 minute. Add 1 ml LB, and incubate 3/4 to 1 hr at 37ºC. I usually plate the whole transformation after spinning down the cells in a low speed centrifuge at 2000 rpm for 5 minutes. If I ligate 100 ng of blunted dephosphorylated vector to 50 ng of a blunt 500 bp PCR in a 30 µl reaction, I'll usually transform 5 ul into cells and plate out the whole tranformation, I normally see approximately 103 colonies (>90% of which are the proper clone).

Ligase Buffer: [660 mM Tris pH 7.5, 66 mM MgCl₂ , 5 mM ATP (added fresh)]