Griffitts:Modified Eckhardt gel
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Materials
- SBE
- 1-Kb DNA ladder
- 10 mg/mL RNase A
- 0.3% Sarkosyl
- Sucrose
- 100 mg/mL Lysozyme
- PH broth
- 10% SDS
- 1.7-mL microcentrifuge tubes
Buffer preparation
5 mg/mL RNase A (300 μL)
- 25 mg/mL RNase A (in –20°C freezer)
- 275 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)
- 900 μL 1X SBE
- 1 g sucrose (=1%)
- 10 μL 100 mg/mL Lysozyme
- 3 μL 5 mg/mL RNase A
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: Pour the gel in the fridge and allow to set for 30 min.
- NOTE: Add the SDS right before pouring the gel, not before microwaving
NOTE: Use the large-tooth comb
NOTE: Use the 1-Kb size standard ladder
Procedure
- 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 150 μL of chilled 0.3% Sarkosyl
- Centrifuge at maximum speed for 90 seconds
- Carefully pull off the supernatant
- Resuspend in 20 μL of SBE lysis buffer
- NOTE: Do not add DNA loading dye!
- Immediately load into an SBE gel
- Allow to sit 2 minutes.
- Run at 23 V for 5 minutes
- Then run at 100 V for 90 minutes
Staining
- Place gel in 100 μL dH2O
- Add 40 μL 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.