P. larvae PCR Identification

Created a 1% agarose gel ( 0.5grams of agarose + 50 mL of TAE buffer) with two 10 well lanes. Loaded DNA markers and PCR amplicons from 7-8-14. 10uL of DNA sample + 2 uL of EZ Vision Loading Dye were loaded into wells. The markers that were used was Hi-Lo DNA Marker from Minnesota Molecular (Link). The base pair sizes per band of the Hi-Lo markers is shown below:

Hi-Lo DNA Marker

Gel was ran at 130 Volts for 30 minutes. The image of the gel is shown below: (it is technically a picture of a picture of a picture!)

Key: 

Top Lanes- AFB primers
1	2	3	4	5	6	7	8	9	10
Bottom Lanes- ERIC primers
1	2	3	4	5	6	7	8	9	10

Code	Sample
2	Spore Stock A
3	Spore Stock B
4	Spore Stock C
5	Plate B (7-5-14)
6	Plate C (7-5-14)
7	Drop Plate - BHI Treatment (7-3-14)
8	Drop Plate - H2O Treatment (7-3-14)
9	Drop Plate - B.A.D. Treatment (7-3-14)

Lanes 1 and 10 contained the Hi-Lo DNA Markers

Note: The gel electrophoresis rig from the teaching lab was used. There were some difficulties with setting/cooling the gel (it seemed to take quite a bit longer than it should have) and with loading (due to depth perception difficulty in using an unfamiliar rig and possibly removing the comb before the gel was completely cooled). 

The top lanes were PCR amplicons using the AFB primer sets and the bottom lanes contained amplicons using the ERIC primer sets (7-8-14). There wasn't enough space on the gel to put the negative control (DH5a). Several of the amplicons were positive in the top lanes and the band sizes seen correlate very closely to what was expected (1106bp). As mentioned, there was some difficulties with loading and cooling the gel, this was especially evident when I loaded the first few wells in the top lane. It is incredibly odd that the Spore Stock B sample (in lane 3 of the top) did not amplify with the AFB primers, but the plate that was inoculated using Spore Stock B did amplify (in lane 5 of the top)... More interestingly, that there was not P. larvae from the Drop plates in the BAD treatment group (in lane 9 of the top). This may mean that the antimicrobial capabilities of the royal jelly component of BAD killed any present bacteria.

The bottom lanes did not amplify at all in any of the samples using the ERIC set of primers. This most likely means that the PCR parameters need to be adjusted for this set specifically. Because the AFB primer set did amplify, it would be expected to have some amplification using the ERIC set as they are both identifying/genotyping designed primers for P. larvae.

Future:

I am planning on re-running this gel using the remaining amplicons as there was so much trouble with loading and cooling of the gel. It would also be worth looking into other isolated colonies of the BAD treatment group from the "P. larae germination in BAD medium assay" study.

//EWW