What is the recipe for preparing a lysis buffer that can lyse both Gram-positive (Gram+) and fungal cell walls for nucleic acid extraction?

Recipe for a Lysis Buffer for Gram-Positive and Fungal Cell Walls

For effective nucleic acid extraction from both Gram-positive bacteria and fungi, a lysis buffer containing proteinase K in a buffered solution with denaturing agents is recommended. 1

Core Components of the Lysis Buffer

Base Buffer Components

• Prepare a buffering agent such as Tris-HCl (10-50 mM) or ammonium bicarbonate at pH 8.0 to maintain optimal pH for enzymatic activity 1
• Add EDTA (1-10 mM) to chelate divalent cations that could otherwise activate DNases 1

Denaturing and Cell Wall Disruption Agents

• Include a strong denaturing agent such as guanidine hydrochloride (4-6 M) or urea (6-8 M) to disrupt hydrogen bonds and hydrophobic interactions 1
• Add SDS (sodium dodecyl sulfate) at 0.5-1% concentration for membrane disruption and protein denaturation 2
• For enhanced fungal cell wall disruption, include 1-2% Triton X-100 as an additional detergent 3

Enzymatic Components

• Add proteinase K at a final concentration of 0.1-0.2 mg/ml (prepare as a 1:1,000 dilution from stock) immediately before use 1
• Include lysozyme (2-5 mg/ml) to specifically target peptidoglycan in Gram-positive cell walls 4

Stabilizing Agents

• Add polyethylene glycol (PEG) at 5-10% to serve as both an osmotic stabilizer and to enhance lysis efficiency 4, 5
• Include sucrose (10-20%) to create osmotic pressure that helps detach the plasma membrane from cell walls 5

Preparation Protocol

1. Prepare the incomplete lysis buffer (can be stored at 4°C):

   • 50 mM Tris-HCl (pH 8.0)
   • 10 mM EDTA
   • 0.5% SDS
   • 1% Triton X-100
   • 5% PEG (molecular weight 8000)
   • 15% sucrose
   • 4 M guanidine hydrochloride 1, 4

2. Immediately before use, prepare complete lysis buffer by adding:

   • Proteinase K to a final 1:1,000 dilution (typically 0.1-0.2 mg/ml)
   • Lysozyme to a final concentration of 2-5 mg/ml 1

Lysis Procedure

1. Add the complete lysis buffer directly to washed cell pellets or samples (typically 50 μl for a 96-well plate format) 1
2. Incubate at 37°C for 60 minutes to allow enzymatic digestion of cell walls 1
3. For particularly resistant fungal samples, consider a pre-solubilization step with SDS followed by sonication 3, 2
4. For Gram-positive bacteria with thick cell walls, mechanical disruption with glass beads may be combined with the chemical lysis for improved efficiency 2
5. After incubation, inactivate proteinase K by heating at 80°C for 30 minutes 1

Critical Considerations

• The lysis buffer volume may need adjustment based on cell density and type 1
• For fungal cells, which have particularly resistant cell walls, a combination of chemical and mechanical disruption often yields better results 2
• Supplementing bacterial growth media with L-threonine or L-lysine before harvesting can make Gram-positive cells more susceptible to lysis 4
• Fresh preparation of the complete lysis buffer (with proteinase K added) is essential for optimal enzymatic activity 1
• The resulting lysate can be used directly as a PCR template without further purification steps 1

Quality Control

• Include both positive and negative controls when processing samples to verify lysis efficiency 1
• For particularly difficult samples, verify DNA yield through gel electrophoresis or spectrophotometric measurement 1
• If working with environmental samples, this buffer composition has been shown to be effective across various sample types 6