Griffitts:Modified Eckhardt gel

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Contents

Materials

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

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.

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