Method: Preparation of Bacteriophage Lambda Concatamers for use as Size Standards in Pulsed-Field Gel Electrophoresis

May 26, 1990

Jim Howe

Purpose:

To produce molecular length size standards accurate in the range of 50-1000 kb.

2 days

Special Reagants:

Low gelling agarose (Seaplaque)

Procedure:

Days 1-2

1. Premelt a mixture of 1% low gelling agarose dissolved in 20 mM MgCl2, 100 mM Tris-HCl (pH 7.6); cool the solution to 37 degrees C.
2. Add 10 µg of high quality bacteriophage lambda DNA to 250 µl of a solution consisting of 2% polyethylene glycol (PEG 8000), 2 mM ATP, and 2 mM dithiothreitol. Add 3 Weiss units of T4 DNA ligase to the mixture, then add 250 µl of the cooled agarose from step 1. Mix gently, then distribute to plug molds to the CHEF DR apparatus (this volume will yield 2-3 plugs; scale up reaction as needed).
3. Allow plugs to solidify on ice for 15 minutes, then remove and place in a 6 well Corning plate with 5 ml of ligation buffer. Seal the plate with parafilm, and allow to incubate at room temperature for 24 hours. Plugs can then be stored in 50 mM EDTA (pH 8.0) at 4šC for up to 3 months. It is useful to test standards on a CHEF gel to ensure their quality before use as standards. A 24 hour run at 200V with a 60 second switching for 15 hours, followed by 9 hours at 90 seconds should reveal a ladder of up to 20 bands on ethidium staining.

Solutions:

• Ligation buffer:

  1 mM ATP
  1 mM DTT (dithiothreitol)
  10 mM MgCl2
  50 mM Tris-HCl pH 7.5
  1% polethylene glycol (M 8000)

References:

Vollrath, D., and R.W. Davis. (1987) " Resolution of DNA molecules greater than 5 megabases by contour-clamped homogenous electric fields." Nucl. Acids Res., 15:7865.