How to Make and Use Common DNA Extraction Buffers

Effectively Lyse Your Cells and Protect Your DNA

DNA can be extracted from cells using a variety of lysis buffers (and sometimes require mechanical methods). Reagents often used in lysis buffers include Tris, EDTA, SDS, CTAB, Triton X100, MgCl₂, KCl, NaCl and other detergents.

With the right DNA extraction buffers and method, you’ll reliably

• Lyse the cellular and nuclear membranes
• Maintain the pH during extraction
• Preserve the integrity of the DNA and protect against degradation
• Separate DNA from cell debris and contaminants

The appropriate lysis buffer recipe depends on your sample type, but for certain applications that are particularly sensitive to contamination, you might want to consider using a reliable DNA isolation kit or ready-to-use DNA extraction buffers.

Also, note that bead beating using lysing matrices is frequently coupled with lysis buffers to increase efficiency and maximize DNA yield.

Plant Sample DNA Extraction Buffers

CTAB based extraction buffers are commonly used for purifying DNA from plant tissue because it facilitates the separation of polysaccharides, and adding polyvinylpyrrolidone can help remove polyphenols.

Reagents & Recipes

• CTAB Extraction Buffer (pH 8)
  • 100 mM Tris-Cl pH 8.0
  • 20 mM EDTA pH 8.0
  • 1.4 M NaCl
  • 2% (w/v) CTAB (cetyltrimethyl ammonium bromide)
  • 1% PVP 40,000 (polyvinylpyrrolidone)
  • 0.3% 2-β-Mercaptoethanol (add directly before use in a fume hood)
• Chloroform: isoamyl alcohol (24:1 v/v)
• 6 M NaCl
• 3 M potassium acetate
• Ice cold 100% isopropyl alcohol
• 70% ethanol

• Rehydrate in 1× TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0, autoclaved)

Read the full protocol.

Plasmid DNA Extraction Buffers

The isolation of plasmid DNA from E. coli using an alkaline lysis is a well-established method. Plasmid-containing bacteria are lysed with a SDS/NaOH solution. Using concentrated potassium acetate causes proteins and chromosomal DNA to precipitate while the plasmid DNA remains in solution and can be isolated using a silica spin column.

Reagents & Recipes

• Resuspension buffer
  • 50 mM Tris HCl, pH 8
  • 10 mM EDTA
  • 100 µg/ml RNase A
• Lysis buffer
  • 0.2 N NaOH
  • 1% SDS
• 3/5 M Potassium acetate, pH 6 (Neutralization buffer)
• Isopropanol
• 70% Ethanol
• Rehydrate/elute in water or TE buffer

Samples with a diverse or unknown bacterial composition—such as for metagenomic—are more complex. Read more about metagenomic challenges and how they are being addressed.

Mammalian Cell DNA Extraction Buffers

Mammalian cell lysis buffer is often used to prepare the cells prior to application on a commercial spin column for DNA isolation. The buffer without RNase can be mixed in advance and stored at room temperature.

Recipes & Reagents

• DNA Lysis Buffer
  • 0.1 M EDTA (pH 8.0)
  • 0.5% (w/v) SDS
  • 10 mM Tris-Cl (pH 8.0)
  • 20 μg/ml DNase-free pancreatic RNase (add right before use)

Blood Sample DNA Extraction Buffers

Extract DNA from fresh or frozen human blood.

Reagents & Recipes

• Red Blood Cell lysis Buffer (pH 7.6):
  • 0.155 mol/L NH₄Cl
  • 10 mmol/L KHCO₃
  • 0.1 mol/L EDTA (Na₂)
  • 20 μg/ml DNase-free pancreatic RNase (add right before use)
• CTAB Extraction Buffer (pH 8.0):
  • 1.5 mol/L Tris pH 7.6
  • 0.4 mol/L disodium salt of ethylenediaminetetra acetic acid (Na₂EDTA)
  • 2.5 mol/L NaCl
  • 2% Cetyl trimethyl ammonium bromide (CTAB)
• 10% SDS (Sodium dodecyl sulfate)
• β‐Mercaptoethanol
• Chloroform: Isoamyl alcohol (24:1)
• Isopropanol
• 70% and 90% ethanol

Read the full protocol.

Sperm Cell Lysis and DNA Extraction Buffers

DNA extraction methods used for human somatic cells are ineffective for sperm due to nuclear compaction and DNA integrity in sperm cells.

Reagents & Recipes

• Lysis Buffer (10x): 50 mL
  • 5 ml Tris-HCl 1 M
  • 1 ml NaCl 5 M
  • 2.5 ml MgCl₂ 1 M
  • 41.5 ml H₂O
• 500 µL RNA-Plus solution
• 50 µL proteinase K
• 500 µL chloroform
• 800 µL pure cold ethanol and 40 µL cold 3 M sodium citrate
• 70% ethanol
• Rehydrate in Tris–HCl or ddH2O

Read the full protocol.

Spectrophotometric Quantification of DNA

Measure the DNA yield and purity using a spectrometer (e.g., NanoDrop) at 260 nm, 280 nm, and 230 nm. Depending on the concentration, you may need to dilute an aliquot of your sample for an accurate reading.