CHAPTER 10

Restriction digest

PURPOSE

Once the optimal conditions for producing fragments between 100 and 350 kb are determined, a mass digestion using several plugs is performed. The partially digested DNA from these plugs then can be used as insert DNA in construction of a BAC library.

EXPERIMENTAL PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose plugs containing genomic DNA (see CHAPTER 7); WB-C; H3M; 10X HindIII buffer; HindIII; 70% ethanol (in a spray bottle); microscope slides and coverglasses (slides and coverglasses should be sprayed with 70% ethanol and dried with a Kimwipe prior to use)

­ Note 10.1: Use the same tube of enzyme and same type of buffer used in the test digest!

METHODS:

  1. Transfer 8-16 of the PMSF-treated plugs into a 50 ml polypropylene centrifuge tube. If the plugs have been stored in 70% ethanol, allow them to equilibrate in WB-C as described in CHAPTER 7, Note 7.5 before proceeding. If the plugs have been stored in WB-C, proceed directly to step 2.

    2. ­ Note 10.2: For mass restriction digests, we generally use enough plugs to provide 150-250 µg of DNA. The DNA concentration of the plugs was determined in CHAPTER 5.

  2. Place the plugs into 20 ml of H3M at 4°C for 1 hour.
  3. Place the plugs on 2-3 clean microscope slides. Macerate each plug using clean coverglasses. Place each macerated plug into its own 1.5 ml microcentrifuge tube. Add 1 ml of fresh H3M to each microcentrifuge tube.
  4. In CHAPTER 9 an optimal enzyme concentration for producing fragments of 100-350 kb was determined. Add diluted NEB HindIII to each tube so that the final enzyme concentration in each reaction tube is the same as the optimal enzyme concentration determined in CHAPTER 9. USE THE SAME TUBE OF ENZYME AND SAME TYPE OF BUFFER USED IN THE TEST DIGEST!

    5. ­ Note 10.3: It is generally best to mix some of the HindIII with 1X HindIII buffer to produce a 1:10 or a 1:100 enzyme dilution. Aliquots of the dilution can then be added to the reaction tubes (see CHAPTER 9). Diluting the original enzyme eliminates troubles normally associated with pipetting stock enzyme (e.g., contamination, dealing with the viscosity of the stock solution, having to pipet extremely small volumes of solution).

  5. Gently mix the contents of each tube, and incubate tubes on ice for exactly 1 hour.
  6. Gently tap each tube to mix the contents, and place the tubes in a 37°C water bath. Remove the tubes from the water bath after EXACTLY 20 min and place tubes on ice.
  7. Immediately add 150 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.

    ­ Note 10.4: Tubes can be stored at 4°C overnight, but at a significant risk. It is highly recommended that the first size selection (CHAPTER 11) is started before pausing.

 

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