Griffitts:Modified Eckhardt gel

Materials

• SBE
• 20 mg/mL RNase A
• 0.3% Sarkosyl
• Sucrose
• 100 mg/mL Lysozyme
• PH broth
• 10% SDS
• 1 mg/mL Ethidium Bromide
• 1.7-mL microcentrifuge tubes

Buffer preparation

20 mg/mL RNase A (300 μL)

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

100 mg/mL Lysozyme (1 mL)

• 100 mg lysozyme (in –20°C freezer)
• 1 mL ddH₂O
• Vortex
• Divide into 100 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.

0.3% Sarkosyl (40 mL)

• 40 mL 1X SBE
• 120 mg Sarkosyl

Note: Store at 4°C

SBE Lysis Buffer (1 mL)

Prepare this fresh each time

• 900 μL 1X SBE
• 100 mg sucrose (= 1%)
• 10 μL 100 mg/mL Lysozyme
• 2 μL 20 mg/mL RNase A

Keep on ice

SBE mini-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: Pour the gel in the fridge and allow to set for 30 min. Don't let it go longer than that or the SDS will crash out of solution.
    

NOTE: Use a large-tooth comb

Sample Preparation and Electrophoresis

• Grow bacteria overnight in LB
• Subculture bacteria in LB and grow to an OD₆₀₀ of 0.6
• Put on ice
• Add 150 μL of each culture to 500 μL of chilled 0.3% sarkosyl
  • If you have a culture with an OD₆₀₀ which does not equal 0.6, use the following shortcut: 90/OD₆₀₀ = the number of μL of culture to add to the sarkosyl
• Centrifuge at maximum speed for 90 seconds
• Carefully pull off the supernatant
  • NOTE: Keep tubes on ice until you load them into the gel
• Resuspend in 20 μL of SBE lysis buffer
  • NOTE: Do not add DNA loading dye!
• Immediately load into an SBE mini-gel
• Allow samples to sit in the well for 2 minutes.
• Run at 23 V for 10 minutes (this is the lowest setting on our machines)
  • NOTE: This works best if you run it without the lid
• Increase the voltage to 100 V for 90 minutes

Staining

• Place gel in 100 mL dH₂O
• Add 40 μL 1 mg/mL ethidium bromide
• Place on shaker at 50 rpm for 60 minutes
• Discard ethidium bromide and replace with fresh dH₂O
• Place on shaker at 50 rpm for 10 minutes
• Photograph under UV light

Adapted from Hynes, M. F. et al. (1989) "Direct selection for curing and deletion of Rhizobium plasmids carrying the Bacillus subtilis sacB gene." Gene 15:111–120. PMID 2548927.