Griffitts:Modified Eckhardt gel

Materials

• SBE
• 1-Kb DNA ladder
• 10 mg/mL RNase A
• 3% Sarkosyl
• Sucrose
• Lysozyme
• PH broth
• 10% SDS
• 1.7-mL microcentrifuge tubes

Buffer preparation

10 mg/mL RNase A (300 μL)

• 50 mg/mL RNase A (in –20°C freezer)
• 250 μL ddH₂O
• Vortex
• Divide into 50 μL aliquots
• Store at –20°C

20X SBE (500 mL)

• 500 mL ddH₂O
• 4 g NaOH (=200 mM)
• 3.72 g EDTA (=20 mM)
• pH to 8.0 using boric acid (powder), ~18 g
  

NOTE: Be sure to dilute this to 1X before using.

3% Sarkosyl (1 mL)

• 10 mL ddH₂O
• 300 mg Sarkosyl

Note: Store at 4°C

Lysis Buffer (1 mL)

• 900 μL 1X SBE
• 1 g sucrose (=1%)
• 1.5 μL 10 mg/mL RNase A
• a few crumbs of lysozyme
• QS to volume with 1X SBE

SBE gel preparation

• 0.4 g agarose
• 50 mL 1X SBE
• 2.5 mL 10% SDS
  • NOTE: Add the SDS right before pouring the gel, not before microwaving
    
  • NOTE: Do not add ethidium bromide

NOTE: Use the large-tooth comb NOTE: Use the 1-Kb size standard ladder

Procedure

• Grow bacteria in PH broth to an OD₆₀₀ of 0.5
• Put on ice
• To 100 μL of each culture, add 500 μL of 0.3% Sarkosyl
• Centrifuge at maximum speed for 30 seconds
• Carefully pull off the supernatant
• Resuspend in 20 μL of lysis buffer
  • NOTE: Do not add DNA loading dye!
• Immediately load into an SBE gel
• Run at 5 V for 15 to 30 minutes
• Then run at 120 V for 60 minutes

Adapted from...