P. larvae PCR Identification

Retrieved two primer sets for identifying and genotyping P. larvae via PCR from Integrated DNA Technologies (IDT). Sales Order #10506260.

Primer sets:

Name	Sequence	PCR-product size	Reference
AFB-F	5'-CTTGTGTTTCTTTCGGGAGACGCCA-3'	1106 bp	Dobbelaere et al., 2001 Link
AFB-R	5'-TCTTAGAGTGCCCACCTCTGCG-3'
ERIC1R	5´-ATGTAAGCTCCTGGGGATTCAC-3´	Several amplicons	Versalovic et al., 1994 (Link)
ERIC2	5´-AAGTAAGTGACTGGGGTGAGCG-3´

Primers were reconstituted by adding nuclease free sterile ddH2O relative to their nm concentration.

Reconstituting PCR Primers

Name	nm conc	Volume added
AFB-F	31	310 uL
AFB-R	23	230 uL
ERIC1R	27.3	273 uL
ERIC2	28.5	285 uL

Crude genomic DNA lystaes were created from Spore Stocks A-C, culture plates of Spore Stocks B- C, and isolated colonies from the P. larvae germination in BAD medium assay (H2O, BHI, BAD treatment groups). Total of 8 different samples. Followed Crude Genomic DNA Lysate for PCR protocol to create DNA lysate.

Set up PCR reactions of each sample using the two different primer sets. Created master mixes for each reaction for a final volume of 25uL per reaction.

Master Mix Set-up (per reaction)

21 uL of Platinum PCR SuperMix (Link)

1 uL of forward PCR primer

1 uL of reverse PCR primer

2 uL of crude genomic DNA

25uL TOTAL VOLUME

Created PCR thermocycler parameters via literature review and named the program "AFB". This AFB program will be used for both AFB-F/AFB-R and ERIC1R/ERIC2 sets of primers.

AFB Thermocycler Parameters

1. 95C for 15 minutes

2. 93C for 1 minute

3. 55C for 30 seconds

4. 72C for 1 minute

5. Repeat 2 - 4 x29

6. 72C for 5 minutes

7. 20C for infinite

PCR amplicons were stored at -20C.

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