Abstract: In research the protocol must be consistent, repeatable, easy and cost effective. Currently there are a number of protocols for isolation of DNA from S. aureus using lysostaphin. However, these have some limitations. The proposed protocol can be informative and useful for researchers for extraction of DNA from staphylococcal cells without addition of lysostaphin. As this chemical is quite costly and increases the cost of DNA extractions in general laboratories. The protocol described here is able to extract staphylococcal DNA in sufficient quantity and quality.

Reagents

• 10mM Tris-HCl (pH 8)
• 25 mg/ml of lysozyme
• Lysis buffer (pH 8)
  • Tris-Hcl (50mM)
  • Sodium dodecyl sulfate (SDS) [1% (w/v)]
  • EDTA (100mM)
• Proteinase K (20 mg/ml)
• Phenol: Chloroform (1:1)
• Chloroform: Isoamyl alcohol (24:1)
• Ethanol (95%)
• Sodium chloride (5 M)
• TE buffer

Procedure

The summarized steps are for extraction of DNA from 10 ml bacterial overnight grown culture

1. Harvest cells from the overnight grown culture by centrifugation at 8000 rpm at 10 °C for 15 minutes and discard the supernatant.
2. Wash the pallet twice with normal saline and centrifuge at 8,000 rpm at 10°C for 15 minutes.
3. Add 0.5 ml of 10 mM Tris-HCl (pH 8) and 2.5 mg/ml of lysozyme and incubate at 37°C for 1- 2 hours.
4. Then add 1 ml of lysis buffer (50 mM Tris, 100 mM EDTA, 1% SDS, pH 8) and 1 mg/ml of proteinase-K. Mix gently and incubate at 50°C for 1-2 hours in a water bath.
5. Digestion with proteinase-K is followed by addition of 1 ml of phenol: chloroform (1:1). Mix gently for 2-3 minutes and centrifugation at 10,000 rpm at 4°C for 15 minutes.
6. Transfer the upper layer in a fresh sterile tube and extract with chloroform: isoamyl alcohol (24:1) by the centrifugation at 10,000 rpm on 4°C for 15 minutes and repeat the step again.
7. Take supernatant and add 50 ml of 5 M NaCl and twice volume of 95% of ethanol and leave it till the precipitation is settle down, minimum for 3 hrs to overnight.
8. Then centrifuge at 10,000 rpm on 4°C for 15 minutes and decant the supernatants and dry the pellet.
9. Add 50 ml – 200 ml of TE buffer (depending upon the size of DNA pellet) for re-suspension of DNA and incubate the DNA at 45°C in a water bath for 2-3 hours for the inactivation of DNA degrading enzymes.
10. Check the quality of DNA on 0.8% agarose gel electrophoresis for further studies.

Observations

The gel electrophoresis revealed a good size band without any smear. The quantity obtained with this protocol ranged between 350 ng-500 ng/ml, the variation in amount depended up on the growth of cells.

Conclusion

This is an alternative method for the isolation of DNA from Staphylococcus aureus without use of lysostaphin and can be easily performed.