Detection of P. larvae in Local Honey

From 1-10-15.

Genomic DNA (gDNA) was extracted from the six brothed cultures by using the QIAGEN DNeasy Blood & Tissue Kit. The protocol for pretreatment of Gram-Positive Bacteria was used (enzymatic lysis buffer was previous prepared and used from 10-17-14). The heat block was used for the 37C and 56C incubation steps. gDNA was frozen at -20C until use.

Received primers that were ordered from Integrated DNA Technology recently. The primers are named 8F and 1492R (Turner et al. 1999). These are universal 16S primers that are commonly used for sequencing.

8F

5'-agagtttgatcctggctcag-3'

Tm = 54.3C

MW = 6,148.0

23.7 nm

1492R

5'-ggttaccttacgactt-3'

Tm = 49.4C

MW = 5,784.8

26.7 nm

I resuspended each primer in DNase-free ddH2O according to their nm. Primer 8F was resuspended in 237 uL (23.7 nm) and primer 1492R was resuspended in 267 uL (26.7 nm). Primers were very gently and brielfy mixed by hand in order to ensure thorough suspension. Primers were stored at -20C in my primer box until use.

//EWW