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Unformatted text preview: Genomic DNA (gDNA) was purified according to the method of xxx (xxx) using . Purified gDNA was restriction enzyme- cut using Eco RI or Sma I (GIBCO) and then was electrophoresed and analyzed using a CYBRSafe DNA gel stain (Invirtrogen, Eugene OR) stained 0.7% agarose gel. Plasmid DNA (pDNA) was purified according to the alkaline lysis method (xxx) using . Purified pDNA was restriction enzyme- cut using Eco RI (GIBCO) and then was electrophoresed and analyzed using a CYBRSafe DNA gel stain (Invirtrogen, Eugene OR) stained 1.5% agarose gel. Materials and Methods Polymerase Chain Reaction (PCR) analysis was performed using foreword (5- catattaccgagggcatattacc-3) and reverse (5-gaggacattaggacatt-3) primers for Staphylococcus pseudintermedius enterotoxin se-int (Futagawa-Saito et al. 2004) designed to amplify a homologous 6000-bp portion of the Staphylococcus aureus pathogenicity island 3 (GenBank accession # AF410775), containing the 781-bp the staphylococcal enterotoxin C (SEC) gene. The PCR was performed using the Platinum TM SuperMix (GIBCO BRL Life Technologies, Gathersberg MD) and the PCT-100 TM Programmable Thermal Controller (Watertown MA). Products were electrophoresed and analyzed using a CYBRSafe DNA gel stain (Invirtrogen, Eugene OR) stained 1.5% agarose gel. Growth Media Tryptic soy agar (TSA) 1. In a 1000mL flask - add:- 500mL of dH 2 O- 13.75g of Tryptic soy broth (Difco; Sparks, MD) - 5g dextrose (Sigma; St. Louis, MO) - 7.5 9g agar (Difco; Sparks,MD) 2. Swirl to mix ingredients (agar will not dissolve unless boiled can get messy on an unattended hot plate autoclaving will accomplish its dissolution) 3. Autoclave the flask at 121 o C for 25 minutes 4. Mix the contents of the flask by swirling 5. Allow flask to cool until it is able to be handled without gloves/protection (about 65 o C) 6. Pour TSA in the flask into Petri dishes, only pour enough to cover bottom of the Petri dish Tryptic soy broth with 1% dextrose 1. In a 1000mL flask - add:- 500mL of dH 2 O . 1 Brain Heart Infusion broth (BHI) 1. In a 1000mL flask - add:- 500mL of dH 2 O . LB broth 1. In a 1000mL flask - add:- 500mL of dH 2 O . Buffers Normal Saline = 0.85% NaCl (w/v) = 0.85 gm NaCl /100mL H 2 O [ 144 mM ] Genos Favorite Phosphate-Buffered Saline ( PBS or is that GFPBS) - pH 7.2-7.4 phosphate buffer saline (PBS; 122 mM NaCl, 10 mM Na 2 HPO 4 , 3 mM KH 2 PO 4 , pH 7.4 NaCl - 7.2 gm fw = 58.96 so final concr. = 122 mM Na 2 HPO 4- 1.48 gm fw = 141.96 so final concr. = 10 mM KH 2 PO 4- 0.43 gm fw = 136.1 so final concr. = 3 mM q.s. with dH 2 O 1000 mL 10x Stock Soln of PBS NaCl - 68 gm fw = 58.96 so final concr. = 115 mM Na 2 HPO 4- 14.8 gm KH 2 PO 4- 4.3 gm adjust pH to 7.2-7.4, q.s. with dH 2 O to 1000 mL An example of another formulation of the many variations of PBS 0.23 g NaH2P04 (anhydrous) (1.9 mM) 1.15 g N~HP04 (anhydrous) (8.1 mM)1....
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