Detection of P. larvae in Local Honey

From 3-27-15.

I attempted to amplify the 16S gene once again in the five selected P. larvae isolates from the local honey sources and the single isolate recovered from the trachea of a roach. I designed my own thermocycler parameter using the primers 8F and 16S Rev. The annealing temperature of 55.8C was used as it was between the two melting temperatures of the primers.

Thermocycler parameters:

Primer sets 8F and 16S Rev
Program name : 8F + 16S

	Temp	Time
Step 1	95.0C	2.00 m
Step 2	95.0C	1.00 m
Step 3	55.8C	1.00 m
Step 4	72.0C	1.00 m
Step 5	GoTo Step 2, repeat x 24
Step 6	72.0C	5.00 m
Step 7	20.0C	Forever

The amplicons were ran on a 1% gel at 120 volts for 30 minutes (10 uL DNA + 2 uL EZ vision loading dye). Unfortunately, the gel was blank once again (nice ladder though!). It is becoming increasingly likely that there is an issue with either my primers or the thermocycler parameter as the DNA that was used as template has been shown to be used successfully in other PCR reactions. I will re-assess this situation soon.

//EWW