Biochemical Techniques and Instrumentation

Procedure

Extraction of DNA

• Take 25 g of seafood sample, homogenize well and transfer into the 225ml of lactose broth. Incubate at 37^oC for 4 to 8 h for pre-enrichment.
• Take 1 ml of enriched culture in a microfuge tube and centrifuge at 10,000 rpm for 10 min at 4 ^oC.
• Discard the supernatant and add 1 ml of molecular grade water to the cell pellet and again centrifuge at 10,000 rpm for 10 min. This step is repeated twice.
• Then, resuspend the cell pellet in 1 ml molecular grade water and place it in a boiling water bath for 10 min.
• Cool the tube immediately and centrifuge at 10,000 rpm for 10 min.
• The supernatant consists of the crude genomic DNA and is used as a template for PCR. This extract can be stored at -20 ^oC until used.
• For positive control, sub-culture the type culture of Salmonella typhimurium (ATCC 98) in a nutrient broth at 37^oC for 16-18 h.
• Then, extract the genomic DNA from 1 ml of the type culture following the procedure described above.

PCR conditions

Primer used for Salmonella detection is invA (invasion protein gene). Forward and reverse primers are synthesized from OCIMUM Biosolutions, Bangalore. The concentration of the forward and reverse primers is 0.4μg /100 μl. The product size of is 275 bp. Prepare the PCR reaction mixture in the following composition:

DNA template - 2.00 μl

PCR buffer* - 2.50 μl

dNTP mix - 0.25 μl

Primer F - 1.00 μl

Primer R - 1.00 μl

Taq DNA polymerase - 0.25 μl

Molecular grade water - 18.00 μl

*PCR buffer contains 100mM Tris (pH 9.0), 500mM KCl, 15mM MgCl₂ and 1% gelatin

Perform PCR in the thermal cycler under the following conditions:

Initial denaturation - 94^oC for 3 min

Denaturation - 94^oC for 60 sec

Annealing - 60^oC for 60 sec

Extension - 72^oC for 60 sec

Final Extension - 72^oC for 3 min

No of cycles - 35 cycles

Analysis

Analyse the amplified PCR product for the presence of Salmonella invA gene by performing agarose gel electrophoresis. Compare the amplified PCR product with 100bp DNA ladder, observe under UV-Transilluminator and document using the gel documentation system.