CHAPTER 9

Test restriction digest

PURPOSE

Construction of a BAC library requires generation of relatively high molecular weight restriction fragments. Such restriction fragments will serve as inserts in BAC construction. In general, fragments between 100 kb and 350 kb are desirable. To obtain fragments in this size range, the high molecular weight DNA in the agarose plugs must be partially digested with a restriction enzyme. To determine the conditions that yield a maximum percentage of fragments between 100 and 350 kb, a series of partial restriction digests is performed.

EXPERIMENTAL PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose plugs containing genomic DNA (see CHAPTER 7); WB-A; WB-B; WB-C; PMSF; 0.5X TBE; agarose; PFGE Lambda Ladder; aqueous ethidium bromide; H3M; 10X HindIII buffer; HindIII; 70% ethanol (in a spray bottle); 70% ethanol; microscope slides and coverglasses (slides and coverglasses should be sprayed with 70% ethanol and dried with a Kimwipe prior to use); CHEF gel apparatus; regular CHEF gel casting stand; 15-tooth gel comb; UV light box equipped with a camera or image-capture system

METHODS:

  1. Remove the plugs from lysis buffer and place them in a 50 ml tube containing WB-A at 50°C for 1 hour.
  2. Decant the supernatant from the tube containing the plugs and add 50 ml of WB-B. Incubate the tube on ice for one hour.
  3. Decant the supernatant and incubate the plugs in 50 ml WB-C for 30 min.
  4. Remove this buffer and add 50 ml of new WB-C. Add 50 µl of 0.1 M PMSF, mix gently, and place the tube on ice for one hour. The PMSF destroys residual Proteinase K in the plugs.
  5. Repeat step 4.
  6. Decant the solution and add 50 ml of ice cold WB-C. Incubate the tube on ice. After 30 min remove the WB-C and add 50 ml of ice-cold fresh WB-C.
  7. Repeat step 6 two more times (for a total of four post-PMSF washes). These washes remove high concentrations of EDTA from the plugs. EDTA inhibits restriction endonuclease activity. Place the tube in the refrigerator.
  8. Make a 1.0% agarose gel in 0.5X TBE using the small BioRad CHEF gel casting stand and the 15-tooth gel comb (see CHAPTER 6 for details regarding gel preparation and loading). Place the PFGE Lambda Ladder in lanes 1 and 13, respectively.
  9. Transfer three of the plugs ("test plugs") into 10 ml of H3M. Let the test plugs equilibrate in the H3M for 1 hour.
  10. Place eleven 1.5 ml microcentrifuge tubes in a tube rack, and label the tubes with consecutive lower case letters (i.e., label tubes a-k). Add 250 µl of H3M to each tube.
  11. In a 1.5 ml microcentrifuge tube, mix 10 µl of New England BioLabs HindIII (20 units/µl) with 80 µl MBG water and 10 µl 10X HindIII buffer to produce a 2.0 unit/µl HindIII solution (Dilution 1). Add 10 µl of this solution to 80 µl MBG water and 10 µl 10X HindIII buffer to produce a 0.2 units/µl HindIII solution (Dilution 2).

    12. ­ Note 9.1: The restriction enzyme used in the partial digest depends upon the sequence of the polycloning site of the vector. There are three cloning sites (HindIII, BamHI, and SphI) in pBeloBAC11 (the most common BAC vector), but only cleavage at the HindIII and BamHI sites produce 5' overhangs for easy vector dephosphorylation (CHAPTER 3). Consequently, HindIII and BamHI are primarily used to construct BAC libraries. In this and following chapters we describe the use of HindIII in constructing a BAC library. However, BamHI can be substituted for HindIII if desired.

  12. Using undiluted New England BioLabs HindIII, Dilution 1, and Dilution 2, add a specific quantity of HindIII to each of the eleven tubes as directed in TABLE 9.1.
  13. Place tubes a-k on ice.
  14. Using a spatula, transfer the three test plugs onto a microscope slide (wiped clean with 70% ethanol). Using a clean coverglass, cut each plug laterally and transversely to produce four pieces of roughly equal size. There should now be twelve ¼ plug pieces.
  15. Macerate (thoroughly chop) each ¼ plug piece with a coverglass.

    16. ­ Note 9.2: Chopping the plugs does not result in appreciable DNA shearing (Wang and Schwartz 1993).

  16. Using a spatula and a coverglass, place two of the macerated ¼ plug pieces (i.e., ½ a plug) into microcentrifuge tube a. Place each of the remaining macerated ¼ plug pieces in one of the remaining ten tubes (b-k).
  17. Gently mix the contents of each tube and incubate tubes on ice for 1 hour. This allows the enzyme to infiltrate the agarose cubes.
  18. Gently agitate each tube and place the tubes in a 37°C water bath. Remove the tubes from the water bath after EXACTLY 20 min, and immediately place the tubes on ice.
  19. Add 30 µl of 0.5 M EDTA (pH 8.0) to each tube (this inhibits further enzyme activity) and gently agitate tubes to promote contact between the agarose and the EDTA. Keep tubes on ice.
  20. Using the pointed end of a spatula, transfer the undigested macerated ½ plug in tube a into well 2 of the gel. Likewise, transfer the macerated ¼ plug in tube b to well 3, the ¼ plug in tube c to well 4, etc. Seal wells with leftover melted 1% TBE agarose (50°C) as described in CHAPTER 5. Run the gel using the following parameters: buffer temperature = 12°C, volts/cm = 6.0, included angle = 120°, initial switch time = 1.0 sec, final switch time = 40.0 sec, ramping = linear, running time = 18 hours.
  21. Stain, destain, and examine the gel as previously described in CHAPTER 6. A diagram illustrating how the optimal enzyme concentration is determined from the gel is shown in FIGURE 9.1. A photograph showing the results of an actual test digestion is seen in FIGURE 9.2.

    ­ Note 9.3: If the partial digest was successful and you are prepared to do a "large-scale" restriction digest within the next day or so (CHAPTER 10), leave 8-16 plugs in the WB-C and store the remaining plugs in 70% ethanol as described in CHAPTER 7, Note 7.5. If you are not prepared to do a large-scale restriction digest, store all of the plugs in 70% ethanol.

INTERPRETING THE RESULTS

 

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