Colony Cracking: Quick Test for Inserts in Plasmids

E. coli cells can be disrupted in an alkaline solution containing detergent. The lysate contains enough DNA to be detected in a single lane of an agarose gel provided that the plasmid have pUC-derived replication origin. The following protocol is modified from that described in the first version of "Molecular Cloning" (T. Maniatis, E. F. Frisch and J. Sambrook, Cold Spring Harbor Laboratory Press, 1982). (NOTE: I seldom use this procedure these days, since PCR-based method described here is more reliable.)

1. Grow bacterial colonies to a large size (2-3 mm) on an agar medium contining an appropriate antibiotic.

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 buffer**. Load the contents onto an agarose gel. As a control, load the plasmid vector without insert on one lane. (Note: It may be very difficult to apply the cracking mixture into the well because of its high viscosity. Loading the mixtures into empty wells rather than the wells filled with buffer and pouring electrophoresis buffer thereafter may give better result.)

6. After electrophoresis, stain the gel by soaking it for 30 minutes in a solution of ethidium bromide (0.5 microgram/ml in electrophoresis buffer).

7. Under UV-illuminator, plasmid DNA should be visible between E. coli genomic DNA (20-30 kb) and low molecular weight RNAs.

* 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.
** Add the loading buffer just before electrophoresis, since bromophenol blue is rapidly degraded in the alkaline solution.

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