Processing DNA for assigning serotype by Intergenic Sequence Ribotyping (ISR)
To prepare genomic DNA preps from your Salmonella cultures, we use Invitrogen PureLink Genomic DNA Mini Kit (Cat # K182002). Any DNA isolation method can be used. Following is the procedure we follow.
Transfer 1 well-isolated colony from any agar plate to 10ml Brain Heart Infusion (BHI) broth. Incubate at 37C for 16 hrs or longer, depending upon strain, to reach stationary phase.
Transfer 1ml culture from BHI to a sterile eppendorf tube.
Centrifuge at 8000 rpm for 10 min to pellet cells.
Re-suspend in 180ul PureLink Genomic Digestion Buffer.
Add 20ul Proteinase K (20mg/ml).
Incubate at 55C for 1 hour.
Add 200ul PureLink Genomic Lysis/Binding Buffer.
Immediately vortex to mix.
Add 200ul 100% EtOH.
Vortex to mix.
Transfer sample to a PureLink Spin Column.
Centrifuge for 1 min at 8000 rpm.
Remove the spin column to a fresh collection tube (discard collection tube with lysate wash-through in it).
Add 500ul PureLink Genomic Wash Buffer 1 to the spin column and centrifuge for 1 min at 8000 rpm.
Remove the spin column to a fresh collection tube (discard collection tube with Wash Buffer 1 wash-through in it).
Add 500ul PureLink Genomic Wash Buffer 2 to the spin column and centrifuge for 3 min at 8000 rpm.
Remove the spin column (discard the collection tube with Wash Buffer 2 wash-through in it) and place it in a fresh, sterile eppendorf tube for DNA elution.
Add 50l PureLink Genomic Elution Buffer and centrifuge for 1 min at 8000 rpm.
DNA preps should have >20ng/ul DNA. Even less concentrated DNA will probably give good PCR results but I wouldnt try if I didnt have at least 10ng/ul. In case of very low cell density in culture, you could repeat DNA isolation and add as little as 25ul elution buffer to get more concentrated DNA.
We use a BioTek Take3 Micro-Volume Plate for measuring DNA concentrations. A Nanodrop Instrument works in a similar fashion. Add 2ul sample to the plate (or 1.6ul to the Nanodrop pedestal) insert in spectrophotometer (close Nanodrop) and read absorbance. BioTek software (built-in for Nanodrop) will show DNA concentration in ng/ul. Any method of measuring DNA concentration can be used.
The processed DNA is sent with ISR primers for sequencing.