CHAPTER 12

Second size selection

PURPOSE

Though the first size selection gets rid of most of the DNA < 100 kb, some small DNA molecules do get trapped by the longer DNA molecules (this is especially true when the DNA concentration in the plugs is relatively high). The second size selection increases the chance that most of these smaller DNA molecules are eliminated prior to ligation.

EXPERIMENTAL PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose cubes x, y, and z containing size-selected DNA (see CHAPTER 11); 0.5X TBE; agarose; LMP agarose; PFGE Lambda Ladder; ethidium bromide; 70% ethanol (in a spray bottle); scalpel with #11 blade; microscope slides; coverglasses; CHEF gel apparatus; large CHEF gel casting stand; 30-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 large BioRad CHEF gel casting stand and a 30-tooth gel comb. Fill the stand until it is near overflowing. See CHAPTER 6 for additional details regarding gel preparation and loading. Allow the gel to thoroughly solidify.
  2. Using a clean scalpel, construct three "slot wells". Each slot well is made by removing the agarose separating four of the comb-made wells (FIGURE 12.1a). Extend each slot well anteriorly to allow enough room for one of the end products of the first size selection (i.e., blocks x, y, and z). Make sure that the agarose lining the bottom of the wells is removed and that the leading edge of each slot well is parallel to the leading edge of the comb-made wells (FIGURE 12.1a).
  3. Using a scalpel and a ruler as a "straight-edge", cut a large block of agarose out of the upper-central region of the gel. The cuts should be made exactly as shown in FIGURE 12.1a. Remove all agarose from this region. Completely fill the resulting cavity with 1.0% low melting point (LMP) agarose in 0.5X TBE. Allow the LMP agarose to solidify.
  4. Place the PFGE Lambda Ladder in lanes flanking the slot wells.
  5. Place blocks x, y, and z in their original top/bottom orientation (see FIGURE 11.1g) in the three slot wells as shown in FIGURE 12.1a. Add LMP agarose to fill any remaining space in each slot well. Gently move each agarose block back and forth to dislodge any bubbles, and position each block so that it touches the leading edge of its respective slot well.
  6. Seal the wells containing ladders with melted LMP agarose. Allow the LMP 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: 6.0 v/cm, included angle = 120°, initial switch time = 3.0 sec, final switch time = 5.0 sec, run time = 18 hours, ramping = linear.

    10. ­ Note 12.1: The gel is generally run overnight.

  10. Using a ruler as a "straight-edge", cut the gel with a scalpel as shown in FIGURE 12.1b. There will be seven gel pieces (referred to as 1-7 from left to right). Using a scalpel, place identification notches at the bottom of some of the gel pieces as shown. These notches allow easy reassembly of the gel later in the protocol.
  11. Stain and destain the odd-numbered pieces of the gel (i.e., the gel pieces containing the PFGE Lambda Ladder). On a UV light box, realign the stained pieces as shown in FIGURE 12.1c. WEAR EYE AND FACE PROTECTION WHEN USING THE UV LIGHT BOX. While observing the illuminated gel, use the scalpel to make small incisions at 125 kb and 350 kb in each of the stained pieces (FIGURE 12.1c).
  12. Turn off the UV light box.

    13. ­ Note 12.2: Never expose the even-numbered gel pieces to UV light as this will break the size-selected DNA and make it unclonable!

  13. On a piece of clean plastic wrap on a workbench, reconstruct the gel by placing the unstained even-numbered pieces between their flanking odd-numbered stained pieces (FIGURE 12.1d). Extend the incisions at 125 kb and 350 kb on each stained gel piece into adjacent unstained (even-numbered) gel pieces. Place the stained gel pieces aside.
  14. Using a scalpel, coverglass, or razor blade, connect the incisions on each even-numbered gel piece as shown in FIGURE 12.1e. You should now have three unstained gel pieces containing DNA between 125 and 350 kb in length. The DNA from these pieces (referred to from left to right as q, r, and s) will be isolated. Cut piece q twice laterally to yield three blocks of roughly equal size. Do the same for pieces r and s (FIGURE 12.1e). Place the three q blocks in a 50 ml polypropylene centrifuge tube. Likewise place the r blocks in a new centrifuge tube and the s blocks in their own centrifuge tube.
  15. If isolation of DNA from the agarose blocks is to be performed within the next two days, 50 ml of sterile 1X TAE should be added to tubes containing gel pieces q, r, and s. Tubes should be stored at 4°C. If DNA isolation is to be performed at a later date, store the agarose pieces in 70% ethanol at –20°C as described in CHAPTER 7, Note 7.5.
  16. Stain and destain the remaining gel pieces. Based on the notches made earlier, reconstruct the gel on a UV light box. If desired, photograph the reconstructed gel for documentation purposes (see FIGURE 12.2 for an example of an actual reconstructed gel).

 

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