Detection of P. larvae in Local Honey

From 3-8-15.
Also from Tripartite conjugation from 1-7-15.

Ran PCR of several different isolates using the AFB primer sets. Crude genomic DNA was used as template DNA for the reaction. I was running low on PCR super mix and really had to skimp on volumes which may have effected accuracy.

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

There was apparently an issue when loading/running the gel as evident by the smears near the wells. The obvious problem appears to be that there is way too much DNA in the wells. My positive control, P. larvae 9545 in Lane 7 of the bottom row, checked out no problem. It is about the correct size as it should be and has a clear and definite band. However, most of the other bands are not so clear. The top row, containing the AFB amplicons, did not possess such clear and definite bands, despite earlier here and here having nice sharp bands. The roach trachea isolate never amplified with the AFB primers and we suspect it is a Bacillus spp.

The bottom row contained three of the transconjugates that grew after the pMarA tripartite conjugation. Only one of them had a nice band (maybe another in Lane 5?). This may confirm that what grew after the conjugation was indeed P. larvae, however, I'll need to still run a PCR to amplify the KanR transposon to determine if the transposon did indeed transfer to the P. larvae. 

Regardless, I am planning on re-running this PCR using the gDNA that I still have. Just as soon as I am able to locate more PCR super mix.

//EWW