Colony Cracking: Quick Test for Inserts in Plasmids

2. Using a sterile toothpick, transfer a small quantity of the colony to a master plate. Transfer the remainder of the colony to a microfuge tube containing 20 microliters of 50 mM NaOH, 0.5% SDS, 5 mM EDTA (cracking buffer).

3. Incubate the tube at 55 C for 30 min.

4. Vortex vigorously for 1 min.*

5. Add an appropriate amount of loading buffeundefined._Load_the_contents_onto_an_agarose_gel_without_ethidium_bromide._As_a_control~E_load_the_plasmid_vector_without_insert_on_one_lane.~K~Hp~M~2~1~0~Kp~M~2~1~0~06._After_electrophoresis~E_stain_the_gel_by_soaking_for_30_minutes_in_a_solution_of_ethidium_bromide_~A0.5_microgram~Hml_in_either_water_or_electrophoresis_buffer~B.~K~Hp~M~2~1~0~Kp~M~2~1~0~07._Under_UV~Filluminator~E_plasmid_DNA_should_be_visible_between_~Ki~ME._coli~K~Hi~M_genomic_DNA_~A20~F30_kb~B_and_low_molecular_weight_RNAs.~K~Hp~M~2~1~0~Kp~M~2~1~0~0~Kbr_~H~M~2~1~0~undefined At this step, long genomic DNA is cut into smaller pieces of about 20-30 kb. Although the original protocol in "Molecular Cloning" does not contain this step, vigorous vortexing is necessary since long genomic DNA in the lysate is troublesome in loading the sample onto the agarose gel.
~undefined_Add_the_loading_buffer_just_before_electrophoresis~E_since_bromophenol_blue_is_rapidly_degraded_in_the_alkaline_solution.~K~Hp~M~2~1~K~Ha~M~K~Hblockquote~M~Ka~M~2~1~Kp~M~2~1~0