TABLE 3.

Timed protocol for aerobic actinomycete clinical isolate identification by PRA^a

PRA step no.	PRA procedure	Time required (h)
1	Cell harvest, suspension in buffer, heat kill, grind in Mini-Beadbeater	1.5
2	Set up PCRs and program Temp-cycler	1
3	PCR amplification in Temp-cycler with 4°C holding mode	Overnight
4	Preparation of four agarose gels (one for step 5 and three for step 8)	1
5	Gel loading and electrophoresis of PCR products (amplicons)	1.75
6	Set up restriction endonuclease digests (three restriction endonucleases and up to 36 digests)	1
7	Incubation of restriction endonuclease digests	1
8	Gel loading and electrophoresis of restriction endonuclease digests (three gels)	2–2.5
9	Photograph gels, evaluate PRA patterns, and identify clinical isolates	2.25

^aTime required to analyze 12 clinical isolates per procedure.