Detection of P. larvae in Local Honey

From 3-11-15.

Re-ran PCR from 3-11-15 using the AFB primers. The lanes are set up as before. 10 ul of ampicon DNA was combined with 2 uL of EZ vision dye and loaded into the well. The 1% gel was ran for 1.5 hours at ~100 volts. The gel is seen below:

Top:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	8a	2a	22a	63a	Q	R. Trach	None
Bottom:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
	Hi Lo	TransConj	TransConj	TransConj	9545	Neg

Discussion:
The good news is that there were nice clean bands seen, the bad news is that there weren't a lot of them (which there definitely should have been). Even the positive control of P. larvae 9545 didn't have a band, which indicates something is clearly amiss. There appears to be a lot of DNA caught in the wells, which may be an indication I am using too much template DNA. Most of the template DNA I used was from a crude genomic prep (heating whole cells), so it is odd that they would result in such a high quantity of DNA. The entire top row was expected to amplify (except for the R. (roach) Trach (trachea) using these AFB primers as they have be shown to in the past. How odd that only a couple of them did. I seem to be having some trouble with PCR lately, it could be attributed to the fact I haven't done it in so long and have lost some of my finesse. I am planning on running more PCR in the near future, but if I am unable to even get my positive control to amplify I am going to have a hard time. So, I will double check my procedure, perhaps I am missing a crucial step or PCR ingredient, or perhaps my thermocycler program has been altered. I will try this applification again using less template DNA, likely diluted 1:10.

//EWW