Table 2.

Reaction conditions

Method	Reaction components	Thermal conditions
Plasmid construction	1.25 mM dNTP mix (Qbiogene)	94°C 45 s/60°C 30 s/68°C 1 min 30 s
	2.5 U of Platinum Pfx polymerase (Invitrogen)	25 cycles
	5 μl of solution Q (Promega, Madison, WI)
	5 μl of 10× Pfx buffer
	2.5 mM magnesium
	100 pmol/primer
	10 ng of template
	Double-distilled H2O to 50 μl
Digoxygenin-labeled DNA probes	10 μl of PCR DIG labeling mix (Roche Diagnostics)	95°C 1 min/50°C 1 min/72°C 30 s
	2.5 U of Taq Polymerase (Fisher Scientific, Pittsburgh, PA)	30 cycles
	10 μl of 10× Taq buffer A
	1.5 mM magnesium
	100 pmol/primer
	10 ng of template
	Double-distilled H2O to 100 μl
Reverse transcription	10 U of AMV-RT (Promega)	42°C 60 min/75°C 10 min
	4 μl of 5× AMV-RT buffer
	2 pmol of (oligo)dT15
	40 U of RNAse inhibitor (Promega)
	2.5 μg of RNA from heat-shocked COS7
	1.25 mM dNTP mix
	Double-distilled H2O to 20 μl