CHAPTER 11

First size selection

PURPOSE

Now that the DNA in the plugs has been digested, it is important to check and make sure that the DNA is of an appropriate length for BAC library construction. DNA longer than 350 kb and much of the DNA < 100 kb is removed during this first size selection. Conversely, most of the DNA between 100 and 350 kb is sequestered and taken through a second size selection (CHAPTER 12).

EXPERIMENTAL PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose plugs containing partially-digested genomic DNA (see CHAPTER 10); 0.5X TBE; agarose; PFGE Lambda Ladder; aqueous ethidium bromide; 70% ethanol (in a spray bottle); scalpel with #11 blade; CHEF gel apparatus; regular CHEF gel casting stand; 15-tooth gel comb; UV light box equipped with camera or image-capture system

METHODS:

  1. Make a 1.0% agarose gel in 0.5X TBE using the regular BioRad CHEF gel casting stand and the 15-tooth gel comb (see CHAPTER 6 for details regarding gel preparation and loading).
  2. Using a clean scalpel, cut and remove the agarose between 4-6 of the center wells of the gel to produce one large "slot well". Make sure that the agarose lining the bottom of the wells is removed and that the leading edge of the slot is parallel to the leading edge of the wells (FIGURE 11.1a). The plug pieces will be loaded into the slot well. Based on the volume of agarose plug pieces, extend the slot well in an anterior direction so that all of the plug pieces can fit comfortably into the slot.

    3. ­ Note 11.1: We commonly cut gels with a scalpel or razor blade. However, some prefer a coverglass arguing that nucleases may be activated by metal ions from the scalpel/razor blade.

  3. Add a small amount of melted agarose to the slot well so that the bottom of the well is completely sealed. Allow the agarose to solidify.
  4. Using the pointed end of a spatula, transfer the macerated plug pieces from all 24 reaction tubes into the slot well. Press the pieces up against the leading edge of the slot well (FIGURE 11.1b).
  5. Place the PFGE Lambda Ladder in lanes flanking the slot well (FIGURE 11.1b).
  6. Seal the slot well and the wells containing ladder DNA with melted agarose. Allow the agarose to solidify.
  7. Remove the gel from the casting stand, and wipe any agarose off the bottom of the base plate.
  8. Place the base plate and overlying gel in the BioRad CHEF electrophoresis chamber. The unit should contain 2.5 L of fresh 0.5X TBE cooled to 12°C.
  9. Run the gel using the following parameters: volts/cm = 6.0, included angle = 120°, initial switch time = 1.0 sec, final switch time = 40.0 sec, run time = 18 hours, ramping = linear.

    10. ­ Note 11.2: The gel is generally run overnight.

  10. Using a ruler as a "straight-edge", cut the gel with as shown in FIGURE 11.1c.
  11. Stain and destain the two peripheral (flanking) pieces of the gel. On a UV light box, align the flanking gel pieces. Turn on the UV light box.

    12. ­ Note 11.3: Always wear eye and face protection when using the UV light box!

  1. Make small incisions in the flanking gel pieces at 100 kb and 350 kb as shown in FIGURE 11.1d.
  1. Turn off the light box. On a piece of clean plastic wrap on a workbench, reconstruct the gel by placing the unstained "center piece" between the two flanking gel pieces (FIGURE 11.1e). Using a ruler as a guide, cut the center gel piece as shown in FIGURE 11.1f so that you have three gel blocks; a center gel block containing DNA between 100 and 350 kb and two "end pieces".

    2. ­ Note 11.4: Never expose the center block to UV light as this will break the size-selected DNA!

  2. Cut the center gel block transversely at two places to yield three blocks (referred to from bottom to top as "x", "y", and "z") of approximately equal size (FIGURE 11.1g). If desired, stain the end pieces with ethidium bromide, partially reconstruct the gel using the various stained pieces, and photograph (e.g., FIGURE 11.2). The unstained pieces should not be included in the reconstruction. Place blocks x, y, and z beneath a piece of plastic wrap to prevent drying.

­ Note 11.5: Continue with the second size selection (CHAPTER 12) before pausing.

 

Return to CONTENTS

Go on to CHAPTER 12