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

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)
NOTE: Be sure to dilute this to 1X before using.
 * 500 mL ddH2O
 * 4 g NaOH (= 200 mM)
 * 3.72 g EDTA (= 20 mM)
 * pH to 8.0 using boric acid (powder), ~18 g

0.3% Sarkosyl (40 mL)
Note: Store at 4°C
 * 40 mL 1X SBE
 * 120 mg Sarkosyl

SBE Lysis Buffer (1 mL)
Prepare this fresh each time Keep on ice
 * 900 μL 1X SBE
 * 100 mg sucrose (= 1%)
 * 10 μL 100 mg/mL Lysozyme
 * 2 μL 20 mg/mL RNase A

SBE mini-gel preparation
NOTE: Use a large-tooth comb
 * 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.

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.